Commensalism is the association of two organisms, one of which derives benefit while the other is unaffected. These relationships are common in nature and in unexpected environments. A good example of commensalism is the survival of nematodes in the intestine of millipedes. The diversity of life capable of living in such an environment is remarkable, sometimes with as many as eight species of nematodes living in the same region of the intestine. The primary goal of this work is to inspire students to gain the requisite skills to discover new life in host organisms that are readily available, accessible, and, in most cases, inexpensive or free. We have created a laboratory protocol to study the diversity of nematode life living inside the millipede intestine. This exercise is designed to teach students to test hypotheses, use taxonomic keys, dissect a millipede, recover nematodes, record data, and formulate a written conclusion. There is a high likelihood that students will discover new species of nematodes during this exercise. The suggested experimental design will catalyze students to investigate the potential of discovering new life in a backyard organism, and simultaneously ignite curiosity and promote a hands-on approach to the application of the scientific method.

## Introduction

The relationship between millipedes and nematodes is generally considered a commensal symbiotic relationship. Nematodes develop in the intestines of millipedes when they ingest nematode eggs while feeding on organic detritus. Nematodes benefit by obtaining food that has already been ingested and digested by the millipede host, without noticeable harm to the latter. Millipedes rely on bacteria to digest most of the food they consume. Most nematodes living commensally in the millipede gut feed on the intestinal bacteria. However, nematodes in the genus Coronostoma are predators on other nematodes (Phillips et al., 2016).

The primary goals of this inquiry and investigation are to demonstrate the relationships between millipedes and nematodes and to examine the biodiversity of nematode life inside the millipede gut. Shannon's and Simpson's diversity indices are used to obtain a quantitative measurement of nematode diversity inside the millipede intestine. Close examination and careful dissections of the intestine allow students to sharpen their observational skills, examine total nematode loads, and understand the concept of commensalism.

Millipedes can be found in moist environments, living on forest floors, in and under decomposing leaf litter, beneath rocks and logs, and below the bark of decaying stumps. They are abundant and readily encountered in optimal habitats and can be collected by simply picking them up and placing them in a zip-lock bag filled with their natural substrate. Millipedes are macroscopic, nonpoisonous to humans, and docile, being unable to bite, pinch, or sting. Millipedes are good organisms to study because they are easy to collect and either free or inexpensive. Excellent examples of published taxonomic keys to Neotropical millipede orders and families are published in Hoffman et al. (1996) and Golovatch et al. (1995). These keys are useful in distinguishing millipedes at any taxonomic level and will guide students on the external features for ordinal separation. A good key to identify the orders of both male and female millipedes can be located through Chicago's Field Museum at https://www.fieldmuseum.org/sites/default/files/millipedekeyenglish.pdf (Sierwald, 2018), and a wide variety of millipede images can be found at https://ag.tennessee.edu/EPP/Pages/Nadiplochilo/Photo-Gallery.aspx (Shelley, 2018).

For the purposes of this laboratory exercise, large-bodied millipedes belonging to the orders Spirobolida, Spirostreptida, and Polydesmida are recommended because their anatomical features are relatively recognizable. We also recommend that millipedes with a body width of 2 mm or more be used, since research has shown that intestinal nematodes are rarely found in smaller specimens (Phillips & Bernard, 2014). Where nematodes are not found in the intestine of a millipede, the resulting Shannon's and Simpson's diversity indices would be zero, whereas a millipede with nematodes would have a Simpson's diversity index >0 and <1.

Nematodes (phylum Nematoda), or “roundworms,” are among the most ubiquitous organisms on Earth and occupy nearly every biological niche (Maggenti, 1981). Nematodes inhabiting the intestines of millipedes pose no risk to humans. Most nematodes are beneficial to humans and other organisms. They promote nutrient recycling and are important for the overall health of the environment. Given the ecological and environmental importance of nematodes, what exactly are they? Nematodes are bilaterally symmetrical, have a complete digestive tract (mouth, intestinal tract, and anus), contain only longitudinal muscles, and have a cuticle consisting primarily of collagen. Except for their lack of respiratory and circulatory systems, nematodes have the same organ systems as vertebrates. The presence of a vulva and eggs readily identifies females; males are generally smaller and usually have spicules (mating organs). Males are often rare or absent in millipede intestines; therefore, for this inquiry, they can be ignored. Figure 1 shows the generalized morphology of a commensal nematode typically found in North American millipedes.

Figure 1.

Nematode morphology.

Figure 1.

Nematode morphology.

## Key to Females of Common Nematodes Found in Millipede Intestines

A user-friendly key to identify common millipede intestinal nematodes is as follows, and a video demonstration can be seen at https://www.youtube.com/watch?v=Xpkl4hA_F8o.

• 1.

First annule cap-like, much longer than next annule; basal bulb without grinding valve; amphid apertures on small horns protruding from anterior end (Figure 2A) … Coronostoma

• 1′.

First annule variable but not cap-like, width usually similar to next annule; basal bulb with grinding valve; amphid apertures not on horns … 2

• 2.

Esophagus with two parts: thick or thin procorpus and subspherical basal bulb with a grinding valve, the two parts not separated by slender isthmus (Figure 2B–E) … 3

• 2′.

Esophagus with three parts: enlarged procorpus, slender isthmus, and basal bulb (Figure 2F, G) … 6

• 3.

Esophagus with thick, muscular procorpus and basal bulb (Figure 2B) … Rhigonema

• 3′.

Esophagus with long, slender procorpus and basal bulb … 4

• 4.

Anterior end of female highly ornate with cuticular collar spines, head with deep grooves lined with rows of scale-like structures (Figure 2C) … Heth

• 4′.

Anterior end of females without spiny ornamentation … 5

• 5.

Head end tapering, rounded or flat anteriorly (Figure 2D) …Thelastoma

• 5′.

Head end mushroom-like, first annule wider than second annule (Figure 2E) … Stauratostoma

• 6.

Isthmus longer than basal bulb (Figure 2F) … Aoruroides

• 6′.

Isthmus shorter than basal bulb (Figure 2G) … Aorurus

Figure 2.

Morphological characters used in the key: (A) Coronostoma, (B) Rhigonema, (C) Heth, (D) Thelastoma, (E) Stauratostoma, (F) Aoruroides, and (G) Aorurus.

Figure 2.

Morphological characters used in the key: (A) Coronostoma, (B) Rhigonema, (C) Heth, (D) Thelastoma, (E) Stauratostoma, (F) Aoruroides, and (G) Aorurus.

## Learning Objectives

• Define commensalism.

• Explain why millipedes are good organisms to study.

• Identify taxonomic groups of millipedes and nematodes.

• Prepare a data sheet (e.g., in Microsoft Excel) showing total nematode loads, taxonomic grouping (species), and the frequency of species.

• Form a null and alternative hypothesis about the biodiversity of species living inside the millipede intestine.

• Calculate Shannon's diversity index and equitability.

• Calculate Simpson's diversity index.

• Write a short laboratory report including the following headings: synopsis, results, data analysis, and conclusion.

## Class Schedule to Complete Project

Two days are needed to complete this laboratory inquiry, with each classroom period lasting 90–120 minutes. Approximately 2 hours will be needed to complete the report, including data analysis, results, and conclusions.

The Next Generation Science Standards (NGSS) recommend that students have the ability to ask questions; plan investigations; collate, analyze, and interpret data; use mathematics to support scientific claims; articulate explanations; and effectively communicate findings to peers (NGSS Lead States, 2013). The National Research Council (2012) recommends that K–12 students be introduced to the concepts of a three-dimensional approach that focuses on scientific and engineering practices, crosscutting concepts, and core ideas in specific disciplines. Application and implementation of both NGSS and National Research Council standards can be applied and supported through this laboratory inquiry that focuses on millipedes as organisms to demonstrate commensalism. Both NGSS and National Research Council concepts are outlined in Table 1.

Table 1.
Selected recommendations from Next Generation Science Standards (NGSS; NGSS Lead States, 2013) and K–12 Framework for Science Education: Practices, Crosscutting Concepts, and Core Ideas (NRC; National Research Council, 2012) addressed by this activity.
Science & Engineering PracticesDisciplinary Core IdeasCrosscutting Concepts
NGSS: HS-LS2-1: Using Mathematics and Computational Thinking:
Use mathematical and/or computational representations of phenomena or design solutions to support explanations.
NGSS: LS2.A: Interdependent Relationships in Ecosystems: Ecosystems have carrying capacities, which are limits to the number of organisms and populations they can support (HS-LS2-1 and HS-LS2-2). NGSS: Cause and Effect: Empirical evidence is required to differentiate between cause and correlation and make claims about specific causes and effects (HS-LS2-8 and HS-LS4-6).
NGSS: HS-LS2-6: Engaging in Argument from Evidence: Evaluate the claims, evidence, and reasoning behind currently accepted explanations or solutions to determine the merits of arguments. NGSS: LS2.C: Ecosystem Dynamics, Functioning, and Resilience: A complex set of interactions within an ecosystem can keep its numbers and types of organisms relatively constant over long periods of time under stable conditions (HS-LS2-2 and HS-LS2-6). NGSS: Stability and Change: Much of science deals with constructing explanations of how things change and how they remain stable (HS-LS2-6 and HS-LS2-7).
NRC standards:
3. Planning and carrying out investigations
4. Analyzing and interpreting data
5. Using mathematics and computational thinking
6. Constructing explanations
7. Engaging in argument from evidence
NGSS: LS4.C: Adaptation: Changes in the physical environment, whether naturally occurring or human induced, have thus contributed to the expansion of some species, the emergence of new distinct species as populations diverge under different conditions, and the decline – and sometimes the extinction – of some species (HS-LS4-6). NRC standards:
3. Scale, proportion, and quantity
6. Structure and function
7. Stability and change
Science & Engineering PracticesDisciplinary Core IdeasCrosscutting Concepts
NGSS: HS-LS2-1: Using Mathematics and Computational Thinking:
Use mathematical and/or computational representations of phenomena or design solutions to support explanations.
NGSS: LS2.A: Interdependent Relationships in Ecosystems: Ecosystems have carrying capacities, which are limits to the number of organisms and populations they can support (HS-LS2-1 and HS-LS2-2). NGSS: Cause and Effect: Empirical evidence is required to differentiate between cause and correlation and make claims about specific causes and effects (HS-LS2-8 and HS-LS4-6).
NGSS: HS-LS2-6: Engaging in Argument from Evidence: Evaluate the claims, evidence, and reasoning behind currently accepted explanations or solutions to determine the merits of arguments. NGSS: LS2.C: Ecosystem Dynamics, Functioning, and Resilience: A complex set of interactions within an ecosystem can keep its numbers and types of organisms relatively constant over long periods of time under stable conditions (HS-LS2-2 and HS-LS2-6). NGSS: Stability and Change: Much of science deals with constructing explanations of how things change and how they remain stable (HS-LS2-6 and HS-LS2-7).
NRC standards:
3. Planning and carrying out investigations
4. Analyzing and interpreting data
5. Using mathematics and computational thinking
6. Constructing explanations
7. Engaging in argument from evidence
NGSS: LS4.C: Adaptation: Changes in the physical environment, whether naturally occurring or human induced, have thus contributed to the expansion of some species, the emergence of new distinct species as populations diverge under different conditions, and the decline – and sometimes the extinction – of some species (HS-LS4-6). NRC standards:
3. Scale, proportion, and quantity
6. Structure and function
7. Stability and change

## Experimental Design

### Materials

• Millipedes can be easily collected in areas where there is moist soil and around streams and creeks, under rocks and logs, and under bark. Larger millipedes are more active in late spring, summer, and fall in areas where there is sufficient moisture. In urban areas where there is little to no natural area, they can be purchased from biological supply houses such as Carolina Biological Supply (https://www.carolina.com/millipedes-and-centipedes/millipede/FAM_143124.pr), Fisher Scientific (https://www.fishersci.com/shop/products/carolina-millipedes-2/p-4655726#?keyword=millipedes), or through private dealers (xericbayou@gmail.com).

• Gloves and goggles

• One large millipede per group of two to four students

• Ethyl acetate or chloroform (optional anesthetizing agents)

• Distilled water, water bottle

• Forceps, minuten pins, dissecting probe, Syracuse watch glass or small plastic Petri dish

• Bright-field dissecting microscope

### Supervised Inquiry Activity

Activity class, Day 1 (90–120 minutes):

• Divide students into working groups of two to four.

• Understand keys to identify millipedes and nematodes.

• Discuss general millipede and nematode anatomy.

• Provide students with live specimens of large millipedes. Explain handling techniques and defensive mechanisms of millipedes. Allow students to handle millipedes prior to dissections (wear gloves and goggles when handing live millipedes).

Activity class, Day 2 (90–120 minutes):

• Gather students into groups of two to four. Students must put on gloves before handling millipedes, whose natural defense mechanism releases pungent chemicals from their defense-gland openings (ozopores) to deter predators; some of these chemicals have the potential to stain and irritate human skin and eyes; however, there has never been an instance of people being seriously harmed by millipedes. If the millipede secretions do come into contact with skin or eyes, use an eyewash station to rinse eyes, and soap and water to clean exposed skin. Do not taste millipedes!

• Millipedes can be anesthetized using chloroform or ethyl acetate prior to dissection, or they can be decapitated with a razor blade. If using anesthesia, while under a fume hood, place two or three drops of chloroform or ethyl acetate in an enclosed container and place the millipede in the container for 5 minutes or until there is no observable movement. Alternatively, if anesthesia is not available, hold the millipede down on a flat surface and remove the head with the razor blade, then loosen the last one or two posterior-most segments and firmly pull the loosened segments, which will extract the intestine (see video for demonstration at https://www.youtube.com/watch?v=ssCMJhZcJ5w).

• Place the removed intestine in a Syracuse watch glass or small Petri dish filled with distilled water.

• Using a pair of fine-tip forceps, gently pull the intestine apart, thereby exposing the inner contents.

• Once the nematodes are observed, gently squirt distilled water on the dissected intestine to dislodge them or gently remove them with a minuten pin or a probe.

• Under the dissection microscope, sort into taxonomic categories (e.g., Coronostoma, Heth, Rhigonema, Thelastoma, Stauratostoma, Aorurus, Aoruroides, and unknown). The shape of the head and esophagus determines the taxonomic ranking (genus or species). Count the number of nematodes for each genus and record on data sheet (for bright-field and scanning electron microscopy images of these genera, refer to plates 1 and 2 at http://epp.tennessee.edu/people/directory/dr-ernest-c-bernard/).

• Discard the millipede carcass into a plastic bag or place it back in its natural environment for decomposition. Nematodes can be preserved on permanent glass slides using methods designed by Seinhorst (1959), stored in 95% ethyl alcohol, or discarded down the sink.

• Clean work station, wash hands with warm soapy water, and work on the lab report.

• Discuss findings within groups, analyze data, determine Shannon's and Simpson's diversity indices, calculate equitability, and share results with the class upon completion of the lab report.

• Grade the lab report and offer constructive comments.

### Data Collection

Students should record their data in a spreadsheet or a lab notebook (we recommend using Excel since the calculations will be fairly straightforward). Once the nematodes have been removed, sorted, and taxonomically classified, students should record the number of each species. After obtaining frequency counts, Shannon's and Simpson's diversity indices can be easily calculated. To calculate Shannon's diversity index and equitability, the following equations are applied:

$Equitability=E=H/Hmax$

where H = Shannon's diversity index, pi is the proportion of nematodes of one genera or species divided by the total number of nematodes removed from the millipede intestine, ln is the natural log, ∑ is the sum of the calculations, and Hmax is the theoretical maximum nematode diversity contained in each millipede intestine. Equitability (E) is a measure of how close the observed diversity is to the theoretical maximum diversity. The closer the equitability value is to 1, the more diverse the intestinal nematode community. A high Shannon's diversity index number suggests that the observed species are evenly distributed and are a diverse community. An example using nematode data extracted from a polydesmid millipede, Apheloria montana, is shown in Table 2.

Table 2.
Data in Excel format showing nematode frequencies from the polydesmid millipede Apheloria montana, where pi is species frequency divided by the total sum (e.g., 2/198 = 0.0101), pi(ln pi) is the product of pi and ln pi (e.g., 0.0101 × ln 0.0101 = −0.0464), and H = −∑ pi(ln pi) = 0.6854.
SpeciesFrequencypipi(ln pi)
Aoruroides sp. 0.0101 −0.0464
Thelastoma sp. 1 0.0051 −0.0267
Thelastoma sp. 2 10 0.0505 −0.1508
Thelastoma sp. 3 0.0354 −0.1182
Rhigonema sp. 164 0.8283 −0.1560
Stauratostoma sp. 14 0.0707 −0.1873
Total no. of species = 6
∑ 198  H = 0.6854
Hmax = ln 6 = 1.791
Equitability = H/Hmax   E = 0.3827
SpeciesFrequencypipi(ln pi)
Aoruroides sp. 0.0101 −0.0464
Thelastoma sp. 1 0.0051 −0.0267
Thelastoma sp. 2 10 0.0505 −0.1508
Thelastoma sp. 3 0.0354 −0.1182
Rhigonema sp. 164 0.8283 −0.1560
Stauratostoma sp. 14 0.0707 −0.1873
Total no. of species = 6
∑ 198  H = 0.6854
Hmax = ln 6 = 1.791
Equitability = H/Hmax   E = 0.3827

Simpson's diversity index is a quantitative measure of dominance of one species over another in an ecosystem, in this case within the gut of a millipede intestine. Another way to describe Simpson's diversity index is as the probability that two randomly selected individuals within the sampling unit (the gut of the millipede) will belong to a different species. It also takes into account species richness (the number of different species) and evenness (how equally the species are distributed within an ecosystem), with a range of values between 0 and 1. An index of 0 indicates no diversity, and values approaching 1 have greater biodiversity. To calculate Simpson's diversity index, the following formula is applied:

$D=1−∑i=1s(n/N)2$

where D is Simpson's diversity index, N is the total number of nematode species present in the sampling unit (millipede intestine), s is the number of species, n is the number (frequency count) of individual species, and ∑ is the sum of the calculations. The range in values for D will be between 0 and 1, where values near zero show low diversity and evenness and values approaching 1 indicate higher biodiversity and evenness. An example of Simpson's diversity index can be seen in Table 3.

Table 3.
Simpson's diversity index showing six species of nematodes extracted from a polydesmid millipede, Apheloria montana, where (n/N)2 is the frequency of nematodes from one species divided by the total frequency of all species, then squared (e.g., (148/169)2 = 0.7669), and summed and subtracted from 1 (e.g., D = 1 − 0.7751 = 0.2249).
SpeciesFrequency (n)(n/N)2
Rhigonema sp. 148 0.7669
Stauratostoma sp. 15 0.0079
Thelastoma sp. 1 0.0001
Thelastoma sp. 2 0.00003
Aorurus sp. 0.0001
Coronostoma sp. 0.00003
∑ 169 0.7751
D = 1 − ∑  0.2249
SpeciesFrequency (n)(n/N)2
Rhigonema sp. 148 0.7669
Stauratostoma sp. 15 0.0079
Thelastoma sp. 1 0.0001
Thelastoma sp. 2 0.00003
Aorurus sp. 0.0001
Coronostoma sp. 0.00003
∑ 169 0.7751
D = 1 − ∑  0.2249

## Student Assessment (Potential Questions)

1. What hypotheses can you develop concerning the biodiversity of nematodes living inside the gut of millipedes? For example, there will be no significant difference in nematode abundance when comparing nematofauna from male and female millipedes. What type of statistical test can you think of that would test this hypothesis?

2. What is the most dominant nematode species found in the millipede gut that you dissected? Offer an explanation why one species may be more dominant than the other.

3. What would you expect the Shannon's and Simpson's index numbers to be if all species were equally abundant? What if they were skewed (e.g., one species is much more dominant than the others)? Explain.

4. What types of experiments can you design to show that different treatments can produce different effects on nematode diversity within the intestine of a millipede? For example, varying the millipede's diets may increase or decrease the nematode biodiversity.

## Conclusion

This lab exercise was initially demonstrated to students varying in age from 13 to 17 as part of a workshop at the Great Smoky Mountain National Park Research Center in 2013. Between 2013 and 2018, several other demonstrations and lab exercises were conducted with Knox County, Tennessee, schools as well as with students from Harriman Middle School, Harriman, Tennessee. Most students were very enthusiastic about the lab and indicated that it was one of their favorite practical exercises. Most of the students indicated that the least favorite aspect of the exercise was decapitating the head of the millipede, so it is suggested that the instructor give the students the option of removing the head or having the instructor do it for them. The vast majority of students were fascinated with the diversity of nematodes living commensally inside the millipede intestine. Many students had never heard of a nematode before this lab exercise, and few were able to describe the difference between a millipede and centipede. The use of millipedes to demonstrate commensal relationships is a cost-effective and easy way to engage students in the classroom and, at the same time, provide a strong foundation in fulfilling the Next Generation Science Standards and the National Research Council's (2012) K–12 Framework for Science Education: Practices, Crosscutting Concepts, and Core Ideas. Close examination and attention to detail provide an excellent opportunity for students to identify new species of nematodes and other commensal organisms. If students and teachers believe that they have found a new species of nematode, the authors would gladly provide guidance and advice to publish their results. The excitement and thrill of being the first to discover a new life form is a monumental experience for any scientist. Students willing to take the time to look for new life, report their findings in a peer-reviewed journal, and establish professional reputations will ultimately attain significant academic and personal successes.

The authors thank all the students who participated in this inquiry and investigation, specifically those who attended the Discover Life in America workshop; students from local Knoxville, Tennessee, high schools; students from Anderson County High School, Clinton, Tennessee; and Veronica Gibson from Harriman (Tennessee) Middle School. We are grateful to Claire Phillips for making the YouTube videos and the commentaries. We acknowledge the use of millipede images from the University of Tennessee at Knoxville's Nadiplochilo web page. We are very grateful to the Tennessee State Parks and Director Roger McCoy for issuing permits to conduct research within the state parks. We are grateful to Dr. Petra Sierwald of the Field Museum for providing the millipede key.

## References

References
Golovatch, S.I., Hoffman, R.L., Adis, J. & de Morais, J.W. (
1995
).
Identification plate for the millipede orders populating the Neotropical region South of Central Mexico (Myriapoda, Diplopoda)
.
Studies on Neotropical Fauna and Environment
,
30
,
159
164
.
Hoffman, R.L., Golovatch, S.I., Adis, J. & de Morais, J.W. (
1996
).
Practical keys to the orders and families of millipedes of the Neotropical region (Myriapoda: Diplopoda)
.
Amazoniana
,
14
,
1
35
.
Maggenti, A.R. (
1981
).
General Nematology
.
New York, NY
:
Springer
.
National Research Council
(
2012
).
K–12 Framework for Science Education: Practices, Crosscutting Concepts, and Core Ideas
.
Washington, DC
:
NRC
(
2013
).
Next Generation Science Standards: For States, By States
.
Washington, DC
:
. https://www.nextgenscience.org.
Phillips, G. & Bernard, E.C. (
2014
).
Survey and revision of nematodes inhabiting North American millipedes
.
Journal of Nematology
,
46
,
219
220
.
Phillips, G., Bernard, E.C., Pivar, R.J., Moulton, J.K. & Shelley, R.M. (
2016
).
Coronostoma claireae n. sp. (Nematoda: Rhabditida: Oxyuridomorpha: Coronostomatidae) from the indigenous milliped Narceus gordanus (Chamberlain, 1943) (Diplopoda: Spirobolida) in the Ocala National Forest, Florida
.
Journal of Nematology
,
48
,
159
169
.
Seinhorst, J.W. (
1959
).
A rapid method for the transfer of nematodes from fixative to anhydrous glycerin
.
Nematologica
,
4
,
67
69
.
Shelley, R.M. (
2018
).
The myriapods, the world's leggiest animals
. Department of Entomology and Plant Pathology, University of Tennessee at Knoxville. https://ag.tennessee.edu/EPP/Pages/Nadiplochilo/Photo-Gallery.aspx.