The Secret Lives of Nematodes
Using entomopathogenic nematodes in the classroom
Author: Samantha Orchard [email protected]
Thank you to my thesis advisor, Dr. Heidi Goodrich-Blair, for exposing me to nematodes and their secret lives!
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.
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
"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. "
"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 108-9, actinomycetes 105-8, fungi 105-6, micro-algae 103-6, protozoa 103-5, nematodes 101-2, other invertebrates 103-5×There may be more organisms in a gram of soil that there are human beings on this Earth!"
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.
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):
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
Class periods 2 and 3: Check on insects and brainstorm
©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.
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:
Additional, optional, class periods:
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!
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):
General information on nematodes; not targeted to K-12 but is fairly easy to follow. Has pictures, definitions and information on categories of nematodes
Nematode information targeted for K-12. Has pictures, lifestyle information and a section on insect-pathogenic nematodes
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).
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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