The Secret Lives of Nematodes

Using entomopathogenic nematodes in the classroom

Author: Samantha Orchard samantha_orchard@yahoo.com

Thank you to my thesis advisor, Dr. Heidi Goodrich-Blair, for exposing me to nematodes and their secret lives!

Topics:

• Overview of activity
• Introduction to nematodes
• Entomopathogenic nematodes
• Finding entomopathogenic nematodes
• What students can learn from entomopathogenic nematodes
• Links

Overview of activity

Nematodes are small roundworms found in the soil. Different nematode species have different lifestyles, with some causing disease in insects, plants, or even humans. This activity focuses on insect-parasitic (entomopathogenic) nematodes. Students will collect soil samples, bait the soil with a living insect larva and monitor what happens in the coming weeks. Some of the insects will die, which presents a "murder mystery" for the students to solve. Typically, insects will die from a variety of causes (e.g. infection by nematodes or fungus or possibly other "natural" causes like hunger/dehydration) and students may only be able to solve the mystery for those insects killed by nematodes. The nematodes, having killed and consumed the insect from inside and reproduced, will leave the insect at some point to search for a new insect to kill and eat. It is at this point that the progeny nematodes can be harvested and visualized with a dissecting microscope. The students can then test their hypothesis that the nematodes killed the insect (if that is their hypothesis!) by using the collected nematodes to try to kill a new insect. This activity requires about 4 class periods (about half an hour each) each spread 1-2 weeks apart.

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Introduction to nematodes

Microscopic images of nematodes; ©Samantha Orchard. Used with permission

Information on nematodes can be found on the Internet. See the "Links" section on this page.

Below are some quotes collected from the Internet about nematodes:

"Nematodes are simple roundworms. Colorless, unsegmented, and lacking appendages, nematodes may be free-living, predaceous, or parasitic. Many of the parasitic species cause important diseases of plants, animals, and humans." --Randy Gaugler

http://www.nysaes.cornell.edu/ent/biocontrol/pathogens/nematodes.html

            "Nematodes are the most numerous multicellular animals on earth. A handful of soil will contain thousands of the microscopic worms, many of them parasites of insects, plants or animals. Free-living species are abundant, including nematodes that feed on bacteria, fungi, and other nematodes, yet the vast majority of species encountered are poorly understood biologically. There are nearly 20,000 described species classified in the phylum [Nematoda].

            "Nematodes are structurally simple organisms... [they] possess digestive, nervous, excretory, and reproductive systems, but lack a discrete circulatory or respiratory system. In size they range from 0.3 mm to over 8 meters. "

http://www.ianr.unl.edu/ianr/plntpath/nematode/wormgen.htm

"If all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable"

N. A. Cobb, 1914

            "It has been estimated that if you look at one 'gram' of soil you will see the following numbers of organisms--bacteria 10^8-9, actinomycetes 10^5-8, fungi 10^5-6, micro-algae 10^3-6, protozoa 10^3-5, nematodes 10^1-2, other invertebrates 10^3-5×There may be more organisms in a gram of soil that there are human beings on this Earth!"

http://nematode.unl.edu/gramsoil.htm

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Entomopathogenic Nematodes (EPN's)

"Entomo-" = Insect

"pathogenic" = Producing disease

            Entomopathogenic nematodes are nematodes capable of infecting and killing insects. They are found free-living in the soil, where they are in a non-feeding juvenile stage. Some species of entomopathogenic nematodes move through the soil in search of a fresh insect larva, while others wait for a larva to crawl by them. Nematodes enter the insect larva through the insect's natural openings: mouth, anus, spiracles (breathing pores). Some species of entomopathogenic nematodes have teeth with which they can break through the insect's hard cuticle and enter directly into the interior of the insect.

            Regardless of how they get into the insect, the nematodes make their way to the hemocoel of the insect. The hemocoel is the body cavity where the insect's organs are bathed in hemolymph, the "blood" of insects. Some entomopathogenic nematodes, while capable of being harmful to insects, enjoy a beneficial relationship with another organism: bacteria. The non-feeding juvenile nematode carries a specific type of bacterium in their intestines. When the nematode reaches the hemolymph, it releases the bacteria it has been carrying. Together, the bacteria and nematode kill the insect larva. In the days following infection, the bacteria break down the insect into its molecular components so that they and the nematode can feed and multiply. The nematode's life cycle includes an adult stage, an egg stage, and juvenile stages. When the nematode and bacteria begin to run out of usable nutrients in the insect, the bacteria again take up residence in the nematode, the nematode stops growing at a non-feeding juvenile stage and the nematode-bacteria pair leaves the insect carcass.

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Finding EPN's

There are some mechanical methods (sieving, centrifugation, floating) for isolating nematodes from the soil, but they require special equipment and do not find just entomopathogenic nematodes. A much simpler isolation method is as follows (brief version... see detailed protocol below):

• Collect a soil sample
• "Bait" it with a living insect larva
• In a period over a couple of weeks, the insects should die, the nematodes will multiply inside, and then emerge from the insect carcass to look for a new insect

Apart from being simple and requiring minimal equipment, this technique has the additional benefit that the nematodes multiply, so you will get out more nematodes than you put in!

Detailed Protocol for finding EPN's

Class period 1: Collect soil and bait with an insect

1. Collect a moist soil sample. This can be done with a hand spade and an old yogurt container. If the soil is reasonably moist and not too hot or cold, you should not need to dig down too deep to get soil with nematodes—often, nematodes can be found in the top few inches of soil. Of course, this opens up a possible research area for students—at what depth are the most nematodes recovered? Does it depend on the season? The moisture content of the soil? When I used this with students, they only took enough soil to fill a quarter to a half of a large yogurt container, and didn’t dig deeper than 2 inches because the soil was fairly moist.
2. “Bait” the soil sample with a living insect larva: using forceps (careful not to crush them) or fingers, put 2-3 insects on top of each soil sample. We use larva ("worms") of the Greater Wax Moth, (Galleria mellonella). These are available from several bait stores (e.g. http://www.wholesalebait.com/ (search for "wax worms") or http://www.johnsonbaitplus.com/ (click on "wax worms")). Upon arrival, the insects can be stored in their shipping container at room temperature (this is best, but only if it's not very hot in the room and the container is kept in a cupboard out of the sun) or in the fridge. It is best to use them within two weeks of receipt and it is not necessary to feed or water them during storage. If they are kept in the fridge, remove them from the fridge about an hour before class and let them warm up to room temperature—they should start wiggling and crawling if they are reasonably “fresh,” but even if they don't wiggle, they're fine to use. It may be harder to convince the students that they are alive though!
3. Cover the container with a lid or saran wrap with small holes for air and put it in the dark at room temperature for about a week. During this time, students can monitor their insects daily, if desired.

Class periods 2 and 3: Check on insects and brainstorm

1. Monitor insects to watch for death. Death may be seen by—lack of movement, lack of response to gentle prodding, color change, growth of fungus on insect etc. Occasionally, insects that don’t die begin to pupate. See picture below for examples of dead versus alive insects.

©Samantha Orchard. Used with permission

Photo of Galleria mellonella. Larva on far right is alive, other three are dead. Larva second from right was killed by Steinernema carpocapsae (nematode)/Xenorhabdus nematophilus (bacterium) pair. Others may have been killed by soil-dwelling microbes (e.g. Serratia) or fungus.

2. If the insects have died, have the students come up with possible explanations and put insects into harvesting containers, as described below. Periodically between class periods, have the students check on the dampness of the paper.

• Use lid only of small (~6cm) petri dish (base is not used). The lid has a slightly larger diameter than the base and so it is used to enable commercial filter disks to fit inside. In addition, the lid is not as deep as the base, and so it fits inside the larger petri dish without raising or touching the lid of the larger petri dish.
• Place a filter or paper disk in the lid of the smaller petri dish. Transfer the dead insect to the paper. Moisten the paper just enough so that it is evenly wet, but not so much that there is excess water. The paper must be moist for the nematodes to emerge.
• Into the base of the larger petri dish, add enough water so that the water touches all sides of the dish (a little less is fine too). Put the lid of the smaller dish, with the insect and paper, into the water in the larger plate. At this point, the water in the larger dish should definitely touch all the sides of the larger dish. Put the lid on the larger petri dish and ensure that the smaller petri dish is not touching the lid of the other one (if it is, try removing some water, or find a different dish).
• Ordering information for harvesting setup:

  “Small petri dish” is 60mm in diameter. Catalogue number 08-757-13A for plastic dish from Fisher Scientific (~$105 for 500 plates, or 1 case). 1-800-766-7000.

  “Large petri dish” is 100mm in diameter. Catalogue number 08-757-12 for plastic dish from Fisher Scientific (~$95 for 500 plates, or 1 case). 1-800-766-7000.

  Filter disk is 60mm in diameter. Catalogue number 09-810B from Fisher Scientific (~$4 for 100, or 1 box). 1-800-766-7000.

  Alternatively, reusable glass petri dishes can be used, and white construction paper or coffee filters can be cut to fit the smaller petri dishes. Or, maybe you could place the insect on paper on the lid of a film canister and float the lid in a small amount of water in a yoghurt (etc.) container. Let us know if you find an inexpensive alternative to petri dishes, so we can share it with others!

View from top (left) and side (right):

©Samantha Orchard. Used with permission

Photo of Galleria mellonella (wax worms) killed by an entomopathogenic nematode. The insects are shown in the "harvesting" aparatus--in this setup, the nematodes will emerge from the insects and move to the water in the larger dish. In this picture, you may be able to see the off-white clumps of nematodes emerging from the insect carcasses. Typically, the nematodes appear to emerge from the mouth or anus of the insects. In setting up the apparatus with insects that had been "bait" on soil, you should use only one insect per dish, not multiple ones as this picture depicts.

Class periods 3 and 4:

1. After 7-14 days, nematodes should begin emerging from the insects. They will look like clumps of off-white matter, or you may not see them at all with the naked eye. Nematodes emerging from insects are attracted to water and should move to the edge of the small dish, wiggle up the side of it, over the top, and back down the other side to reach the water in the larger petri dish. If the paper in the small plate is not kept moist, the nematodes will not move out. You may be able to see the nematodes in the water of the larger dish with your naked eye, if you look very carefully, but it is best to look for them under a dissecting microscope (put the whole setup on the base of the microscope and look in the water).

Additional, optional, class periods:

1. To test the hypothesis that the nematodes killed the insect, repeat the above activity but use the collected nematodes from the water (see class periods 3 and 4, above) instead of the soil. Set up the living insect on a piece of moist paper in a clean petri dish as for harvesting nematodes, above, and drop some of the nematode suspension on the moist paper (using >50 nematodes is probably best). Again, be sure to keep the paper moist but not too wet at all times. At the same time or after the insect dies, put the smaller dish into the larger dish with the moat of water. If all goes well, the nematodes should kill the new insect, multiply, emerge, and migrate to the water again.

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What students can learn from EPN's

            Many people have not heard about nematodes, and many that have may not know much about them. In particular, few people have heard about nematodes that kill insects. Since many of the insect species that nematodes kill are agricultural pests in the larval stage, entomopathogenic nematodes impact our lives by reducing crop damage by insects without the use of chemicals toxic to humans. Apart from learning about the valuable agricultural contributions of nematodes, students can also explore interactions between organisms using the bacterium-nematode-insect partnerships.

            "Koch's Postulates" are "the ground rules of proving whether an agent causes a disease" (http://whyfiles.org/012mad_cow/koch_postulates.html). These rules can be examined using entomopathogenic nematodes and insects in the classroom. Robert Koch was a German microbiologist who helped found the field of Microbiology. He set four conditions that must be satisfied to prove that an agent causes a particular disease. These are:

1.     The agent must be present in every case of the disease;

2.     The agent must be isolated from the host and grown in vitro [in a lab dish];

3.     The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host; and

4.     The same agent must be recovered again from the experimentally infected host.

            The entomopathogenic nematode-insect system can be used to show how Koch's postulates work: the nematodes produce a disease in the larvae that kills the insect. Nematodes can be easily isolated from the dead insects and be kept outside of the insect (actually growing them outside of the insect is a more advanced technique, requiring the capacity to make media for growing bacteria). Applying the stored nematodes to "new" insects will kill the insects, and the nematodes can be recovered from this second insect. This cycle occurs over and over (i.e., the nematode kills the insects, nematodes can be recovered from the insect, the nematodes can kill a new insect, the nematodes can be recovered etc.), thus mostly fulfilling Koch's postulates. Koch's postulates can be fully fulfilled if you grow the nematodes outside of the insect using a special medium and bacteria.

            Even if you do not intend to explicitly teach Koch's postulates, the process described for "Finding Entomopathogenic Nematodes" can be turned into a murder mystery activity that goes through Koch's stipulated steps, above. Students can put living insects onto their soil samples and come back a few days later to find their insect dead. The insect can be portrayed as a murder victim and it is the students' job to find out what killed the insect. When the nematodes emerge from the dead insect, the students will have a suspect in their mystery and when the "suspect" kills a new insect, the students have some pretty good evidence that the suspect was at least capable of killing an insect. In addition, the presence of the nematode at the crime scene (in the soil) might make the case against the nematodes pretty compelling!

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Links

There are plenty of websites on the Internet with general information on nematodes. Try searching http://www.google.com etc. for "introduction nematode" or "entomopathogenic nematodes" for additional pages. EPN's are used as "biological control" agents in gardens and agriculture instead of insecticides--try searching for "nematodes biological control" for more information on this topic. Below are a few of the websites I've come across with general nematode information (I got the quotes above from some of these pages):

http://www.ianr.unl.edu/ianr/plntpath/nematode/wormgen.htm

General information on nematodes; not targeted to K-12 but is fairly easy to follow. Has pictures, definitions and information on categories of nematodes

http://ucdnema.ucdavis.edu/imagemap/nemmap/ENT156HTML/ESINDEX

Nematode information targeted for K-12. Has pictures, lifestyle information and a section on insect-pathogenic nematodes

http://www.nysaes.cornell.edu/ent/biocontrol/pathogens/nematodes.html

General information on nematodes; not targeted to K-12, but is fairly easy to follow. Has information on suppliers of nematodes, in case you would like to buy them rather than finding them in the soil. Discusses "biocontrol" uses of nematodes (to kill agricultural pests without the need for chemical pesticides).

http://www.ucmp.berkeley.edu/phyla/ecdysozoa/nematoda.html

General information on nematodes; not targeted to K-12 but is fairly easy to follow. Details the body structure of nematodes and provides links for phylogeny.

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