Water bears & pillbugs: two invertebrate models that offer authentic opportunities to explore research methods in biology.
Abstract: Biological research entails myriad techniques and considerations for proper experimental design and data collection. The duality of field-based research techniques and laboratory protocols makes introducing this topic to high school and undergraduate college students a challenge. Two invertebrate models that serve as wonderful tools to support an inquiry process to balance techniques in the field and in the laboratory are terrestrial isopods and water bears. Both are easy to collect and rear, are relatively little-known species, and offer the chance for students to work directly with aspects of natural history, ecology, and biology. The 10-step process presented here offers an outline to follow in guiding students through a research cycle in the course of a semester (16 weeks).

Key Words: Tardigrade; Oniscoidea; research methods; inquiry; water bear; terrestrial isopod.
Article Type: Report
Subject: Biology (Study and teaching)
Isopoda (Research)
Methodology
Science experiments
Author: Rife, Gwynne S.
Pub Date: 08/01/2010
Publication: Name: The American Biology Teacher Publisher: National Association of Biology Teachers Audience: Academic; Professional Format: Magazine/Journal Subject: Biological sciences; Education Copyright: COPYRIGHT 2010 National Association of Biology Teachers ISSN: 0002-7685
Issue: Date: August, 2010 Source Volume: 72 Source Issue: 6
Topic: Event Code: 310 Science & research
Product: Product Code: 8522100 Biology NAICS Code: 54171 Research and Development in the Physical, Engineering, and Life Sciences
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 245037779
Full Text: [ILLUSTRATION OMITTED]

The problems inherent in giving students authentic experiences with biological research are common to all biology educators. Time frames are short, money for doing research is limited, and the time needed to supervise students on an individual basis is substantial. In the end, the data collected are seldom suitable for publication.

Over the course of my sabbatical leave, our biology program grew, and it appeared that my class size would double when I returned in fall 2002--and I was already feeling the strain of offering true research experiences to so many students who chose diverse areas in biology as their focus. It was clear that I needed to work toward a full-class project if I was to continue giving the students actual experience with biological research, but I remembered my own undergraduate experience at a larger state school, which was not inspiring. We did a class project with siamese fighting fish and aggressive displays--a great model, but less than exciting to me, as I had observed betas all my life.

I wanted to share a project with my students that was new, and one that the students would have to begin as all research is begun--with some novel observation, followed with a question that would lead to a literature search and end with an investigation. In dwelling on possible projects, I considered topics that might contribute to my own research interests. I had done some preliminary work with two invertebrate models: water bears (Tardigrada) and pillbugs (Oniscoidea). These are lesser-known groups but are abundant in the environment and easy to work with. I have found that they are well suited for giving undergraduates the experience of doing research and creating a robust data set for analysis. These qualities of water bears and pillbugs--that they are available and relatively easy to work with, and that data can be dependably collected--allow enough flexibility in focus and design that students can successfully pursue their own ideas, which offers them the feeling of owning the project. This makes writing up a project in scientific format a challenging experience, and one that cannot easily be copied from another study.

I developed this guided research experience over 15 years, moving from a format in which each student pursued his or her own interests over a semester, with varying results, to a class project in which everyone participated and the results were overwhelmingly positive. Taking a role in designing and executing an experiment and communicating the results in scientific format is key to an authentic experience with the research cycle. Working as a team, the whole class can collect enough data to provide high-quality, if not always conclusive, results.

My doctoral work was with terrestrial isopods (Oniscoidea) in Ohio (Rife, 1993), and I first considered projects using this group. For a fall-semester class, this model offered the perfect opportunity to identify, collect, house, and work with the local species (see Figure 1). However, spring semester presented a problem. Our weather here in northwest Ohio would allow collecting only in the last few weeks of the term, and even then, pillbugs are generally very scarce. Even if pillbugs could be collected, there was no time to complete even the simplest research that would be adequate for written reports.

In considering this problem, I recalled an ecology lab that had been very successful in early March, based on an article that I had read in an issue of The American Biology Teacher about tardigrade diversity (Shofner & Vodopich, 1993). My students had greatly enjoyed the lab, and I had prepared for it by testing sampling methods before the semester began. I quickly learned that water bears offered the perfect model for teaching research methods, and I used them in several fall as well as spring terms. Pillbugs should work equally well for teachers in warmer climates. My labs with both these groups have been satisfying and enjoyable enough to help convince many of my biology majors to continue on to graduate school.

[FIGURE 1 OMITTED]

* Objectives & Benefits

I had clear objectives in developing these labs, for they were used in our senior capstone biology course, our final check that our majors were adequately exposed to the research side of biology in addition to the content they had learned. In designing the course sequence and teaching methodology, being most concerned with making the experience as close to an actual research cycle as possible, I developed the following objectives:

Objective 1: Share the fun and challenges of the field-work portion of biological research.

Objective 2: Provide experiences that require microscope work and an understanding of the economic and physical requirements for high-quality data collection in the biological sciences.

Objective 3: Ensure that the students have the tools needed for using scientific literature to investigate biological phenomena.

Objective 4: Offer an authentic experience that follows the scientific method from design to execution to communication in written form.

Objective 5: Require each student to work individually with a large and robust data set to support or refute a hypothesis that they had a part in developing.

I have found that besides meeting these objectives, this method of teaching offers additional benefits:

* Students must work at the higher level of Bloom's Taxonomy of Educational Objectives (Anderson & Krathwohl, 2001) and use higher-order process skills to successfully complete the work.

* Students discover the dynamics of working in a group and the pros and cons of sharing the responsibility of data collection with others.

* Students become mini-experts on little-known invertebrate species, which can inspire them to be advocates for some of our planet's smaller life forms.

Although all the objectives were not completely met each semester with each student, the formula and sequence presented below have been highly effective. My course met once a week for 3 hours, which made it easy to focus on a week-by-week task list.

* Procedures

The basic procedures are outlined here in a learning cycle of 10 steps, which my classes followed over the 16 weeks of a semester.

1. The students spend a week or two talking about the scientific method and what science is. The instructor can use a variety of activities and inquiries to reinforce the discussion (for one of my favorites, see Favero, 1998).

2. By the third week, the students should be focused on locating scientific literature, be familiar with online literature searches, and be learning the difference between secondary sources that are more familiar to them and primary literature that comes directly from research. At this point, a general lecture on the little-known groups that they will be working with (either water bears or pillbugs) will get the students thinking about these animals and what might be learned in studying them locally.

3. By the fourth week, the students should be familiar with the groups being investigated, should have been reading the current scientific literature on the groups (although some papers will be too technical at this point), and should have taken a walk across campus to collect and observe pillbugs or identify trees with lichen communities to collect water bears from.

4. During weeks 4 and 5, the students work in groups (in my classes, generally five or six groups of four or five students) to choose two possible hypotheses that could realistically be tested by the class (for examples, see Table 1). After much brainstorming and discussion, each group shares their suggested hypotheses, and everyone is instructed to let their scientific impulses work in their conscious and subconscious minds over the next week or two, when the class hypothesis will be selected and the study designed.

5. During weeks 6 and 7, nearing the middle point of the semester, class time involves getting realistic about what can be done. What equipment funds and physical space are available to support this research? What are the bio-ethical issues involved in collecting live animals? How will time and weather present variables that we cannot control? What makes biological research different from research in the other sciences? Groups again meet for discussion, and either the whole class agrees on one hypothesis to test (this was a rare occurrence in my classes; spirited debates were the rule) or two or more hypotheses can be developed and tested by the class, as long as the same data can be collected by all students so that the data set can be used to support or refute each hypothesis, with enough data points for the results to be valuable.

6. In order to allow at least three weeks for data collection, the research-design phase begins as soon as the hypotheses are chosen. All research procedures are designed by the class, with guidance by the instructor to prevent wasting time and resources. It is also important to add discussions regarding the equipment and laboratory space available as part of the field-collection and experimental design process. The instructor can then type out the final agreed-upon procedures, make copies for all the groups, and put together field boxes that the groups can check out, containing the instruments needed for collecting samples.

7. Collection and compilation of data perhaps caused the most angst and confusion in my classes. I refused to be the workhorse. This is a great opportunity for students to experience what it is like to work with biological data and as part of a team. Some keys to success, which should be addressed in the design phase, are that all groups and all group members collect the data, that all data are collected in the same way, and that the data are reported in such a way that they are accessible to all class members. Organization is key to success in working with data.

8. For weeks 10 through 14, while the students are on their own collecting data, the instructor should be available to address complaints about students who are perhaps not pulling their weight, ensure that the groups have access to equipment, and hang out around the lab to help students identify samples when needed.

9. One week before the final week of class, I would set a date when all the data were to be compiled and shared in the form of a massive Excel spreadsheet. From this point on, I became more scarce and let the students wrestle with analyzing the data set on their own to determine whether it supported or refuted the hypothesis. It was common for me to get a frantic e-mail saying that the data set was too big to print out and so the students were lost as to how to begin analyzing it. Reminding them about why we took triplicate samples and how the data would need to be presented in a summarized form usually got them thinking about crunching some numbers before they continued on to analysis.

For the final writing stage, I allowed some deviation from the planned procedure. The data often suggested some very interesting trends that had not been predicted or considered. I allowed the students to write up their project with a new hypothesis, rather than the one chosen by the class, if they determined that this would be a more interesting use of the data. I assigned a textbook that helped them write in scientific form (McMillan, 2006), because the major grade for the course was based on the quality of their final report.

10. In the last weeks of the course, as the research cycle was completed, I waited patiently while the students reached their own "Aha!" moments and met with those who still felt shaky about the writing format. I was always ready for the question "Can we just all turn in the same paper as a group?" I told them that no, at this time everyone was given the same data set and everyone needed to prepare their own paper. I always allowed students to meet with me individually to work over a rough draft, and this was time-consuming, of course, but it yielded such evidence that the process worked and that the class objectives had been met that it was well worthwhile.

The best exchanges came when a student would say something like "Dr. Rife, I see what you mean about graduate school, I think I might like doing research...." Knowing that a student had developed the confidence to consider a graduate program was one of the nicest reinforcements that his method of teaching was right on target. So often, with an inquiry approach, it feels like doing a high dive in the dark. Each time I jumped with this method, I hit the water without a ripple and found it to be warm and satisfying.

Although the actual procedure and data would vary each semester, as the students were directing the details, I always had some baseline procedures and data points that I ensured were part of the experience. I also made sure that enough data were collected to make the analysis likely to yield some trends. For each of the models there were a few important pieces and some major support information needed, and these are outlined in Table 2. Table 3 provides examples of equipment and useful data in the laboratory phase.

* Pillbugs (Oniscoidea): Basic Pros, Cons, & Procedures to Ensure Success

It was easy for me to help the students identify the pillbugs they collected to species, because my doctoral research focused on this group and the Ohio species. I referred them to an online key that I had published or supplied them with written copies. In addition, several good macroinvertebrate references are available (e.g., Pennak, 1978). Pillbugs are generally well known and not difficult to identify, and there is also a great deal of literature on how to culture in the classroom (e.g., Burnett, 1992).

Knowing the species of the research subjects was a clear advantage in using this group, as it made the literature searches and writing of the projects scientific. Although the collecting of pillbugs was dependable early in the semester, a dry period in late fall could prevent further collection if it was needed or planned, so I tended to include a culture for the duration of our project in the campus greenhouse (pillbugs are easy to maintain in a classroom).

The very best hypothesis and set of projects came from a class that tested the maze-running behavior across two species of pillbugs. A T-shaped maze made from Lego blocks was used, with different foodstuffs offered at the ends. The control-group results (where T-shaped mazes had no foodstuffs in either left or right end) worked to illustrate that one of the species was predisposed to turn right, whereas the other tended to hit the far end and then turn most often to the left. On the ethics side, the pillbugs incurred minimal harm from handling and could be released after testing.

* Water Bears (Tardigrada): Basic Pros, Cons, & Procedures to Ensure Success

Water bears are very poorly known, and identification to species was nearly impossible for all the species we collected in my classes. Personal communication with William R. Miller, who has several wonderful publications and online resources, was a crucial help, and I used a modified version of a PowerPoint presentation that he offered online to introduce the students to these amazing animals. Time during the semester generally did not permit identification to species by use of a compound microscope and the preparation of the tardigrades (Miller, 1997; Miller & Case, 1998; W. R. Miller, unpublished data). I have had students who continued their study as independent research or honors projects, and even then not all species were identifiable.

On the positive side, water bears in our area were very distinct from one another. Often they had distinctive pigments: one species appeared as if it were pink-polka-dotted, and another had an opaque golden color. Hence, naming them species A, species B, and so on was a workable way to identify them for data collection and use them for diversity indices. The amazing capabilities and relatively obscure nature of this group made them a superior model. There is very little scientific literature on them, and this obscurity made the students feel that the time they spent could actually yield some new discovery, adding an authentic feel to the project.

Students were often amazed to see these tardigrades appear in the bottom of the dish after being submersed for 24 hours in spring water (we always used finger bowls with spring water alone as the controls). By having the students return to make counts at 48 and 72 hours (and each collected sample in triplicate) gave the opportunity for a robust amount of data that could be manipulated and analyzed for trends in many ways. Since each group tended to sample five trees and take three samples from each tree, a whole class would produce, at the minimum, 90 data points for each 24-hour count, and often a group would sample more trees if their hypotheses seemed to dictate it, for example "Trees in the riparian zone will support more tardigrades than those in the adjacent forest area." For this type of hypothesis, three trees for each condition would be a minimum number sampled.

* Conclusions

After 10 years of collecting data from classes in this way, I anticipated that I would have a data set that would yield a publication or two. Looking over the many directions the classes focused on, and the holes in the data, there are currently some favored hypotheses that could be pursued, but the data are not consistent enough to produce a publication. Looking back, it was a good decision to allow this variation and not predetermine, for my own purposes, the data that the students would collect.

The relative lack of information on pillbugs and water bears is a great advantage for student research. After their data are analyzed and they are moving toward supporting or refuting their hypothesis, the students discover how very little specific information exists that pertains to even a straightforward research project. At first, they may think that an Internet search is all that is needed to get supporting literature for their final write-ups. But they quickly learn that although there may be a lot of sites that share information about pillbugs or tardigrades, there are few that have primary research that can be used to support their conclusions, which means that more library time is needed.

Even more valuable from the learning perspective was identifying the myriad variables that could be involved in drawing any conclusions from this relatively quick study. Very often the data collected were in opposition to what the students had predicted, and, as is common with biological research, they were left with many more interesting questions than answers at the end of the course.

Every scientific write-up generally ends with recommendations for future research. For over a decade, as I graded papers, I was always amazed that each student presented his or her own style, focus, and interest areas at the end of the paper. Although in certain classes the weather failed to cooperate with the collection phase or the cleaning staff dumped out "dirty containers" that were actually samples in process, the results never totally failed to expose the students to authentic research experiences. There were always data to use, even if one group was not able to provide their share. And although not all students embraced the research methods and chose to pursue biology further, I do not recall a student who did not, in the end, enjoy and appreciate the newfound knowledge and the feeling of being a part of the small club of biologists who investigate little-known invertebrates.

DOI: 10.1525/abt.2010.72.6.6

References

Anderson, L.W. & Krathwohl, D.R., Eds. (2001). A Taxonomy for Learning, Teaching, and Assessing: A Revision of Bloom's Taxonomy of Educational Objectives. Boston, MA: Allyn and Bacon.

Burnett, R. (1992). The Pillbug Project: A Guide to Investigation. Washington, DC: NSTA Press.

Case, S. (1998). KanCRN, tardigrades & the International Tardigrade Survey. Slow Walker News, 1, 6-7.

Favero, T. (1998). Double dipping for research: an introduction to the scientific method. American Biology Teacher, 60, 524-525.

Hopkin, S.P. (1991). A Key to the Woodlice of Britain and Ireland. AIDGAP (Aids to the Identification of Difficult Groups of Animals and Plants). Field Studies Council Publication No. 204.

Kinchin, I.M. (1994). The Biology of Tardigrades. London: Portland Press.

McMillan, V.E. (2006). Writing Papers in the Biological Sciences, 4th Ed. Boston, MA: Bedford/St. Martin's.

Miller, W.R. (1997). Tardigrades: bears of the moss. Kansas School Naturalist, 43(3), 1-16.

Miller, W.R. (1998). Tardigrades: bears of the moss. [Online.] Available at http: //pathfinderscience.net/tardigrades/.

Miller, W.R. & Case, S.B. (1998). Tardigrades: in the classroom, laboratory, and on the Internet. Bioscene, 24(3), 3-10.

Pennak, R.W. (1978). Fresh-water Invertebrates of the United States, 2nd Ed. New York, NY: Wiley.

Rife, G.S. (1993). A scanning electron microscopy study of cuticular structures in some Ohio isopods (Oniscoidea: Isopoda: Crustacea). Ph.D. dissertation, Bowling Green State University, Bowling Green, Ohio.

Shofner, M. & Vodopich, D. (1993). Diversity in a hidden community: tardigrades in lichens. American Biology Teacher, 55, 418-423.

GWYNNE S. RIFE is Professor of Biology and Education at the University of Findlay, 1000 N. Main Street, Findlay, OH 45840; e-mail: rife@findlay.edu.
Table 1. Examples of hypotheses generated and
tested by students.

                 Fall Pillbug Study          Spring Water Bear Study

General          What species of             Sugar maple trees will
in Nature        pillbugs are resident       support greater numbers
                 on campus?                  of water bears than other
                                             trees on campus.

More             There are fewer             Lichen patches on north
Specific         pillbugs around the         faces of trees will have
                 foundations of campus       fewer water bears than
                 buildings than in the       those on south faces.
                 leaf litter around
                 the bases of trees.

Highest          The population              The density of
Level            density of Trachelipus      tardigrades and the
                 rathkei is correlated       diversity of the species
                 with the weight of          are correlated with the
                 leaf litter in a square     size of the lichen patch
                 meter of forest.            sampled.

Table 2. Basic data collected and equipment used for field study
and/or collection of samples.

           Identification       Equipment            Data
           References

Pillbug    Online               Thermometer,         Date, time, GPS,
Study      identification       collection vials,    temperature,
           key: http://         forceps (or          relative
           www.geocities.com/   plastic spoons),     humidity,
           ~gregmck/woodlice/   vials with wax       location type,
           ohio_key.htm         pencils to label     soil pH, species
                                date and sample      collected/
           Hard-copy            number, method to    observed
           references           measure relative
           supplied to all      humidity, hand
           in the class:        lens, GPS unit
           Hopkin, 1991;
           Pennak, 1978;
           Rife, 1993

Water      Identification       Thermometer,         Date, time, GPS,
Bear       to family only       paper bags,          temperature,
Study      was used, as this    pencil to label      relative
           group is not well    bags with date,      humidity, tree
           known                sample number,       species, lichen
                                field data           type, soil pH,
           A Web site that      sheets, string       percent lichen
           was very helpful     for tree             coverage,
           in the laboratory    circumference        direction of
           phase: http://       measurement,         lichen patch
           tardigrade.          sharp implements,    faces (N, S, E,
           acnatsci.org/        lichen-coverage      W), height from
                                clear sheet, GPS,    base of tree the
           Hard-copy            tree                 sample was taken
           references           identification       from,
           supplied to all      guide                circumference of
           in the class:                             tree sampled
           Kinchin, 1994;
           Miller, 1997;
           Pennak, 1978

Table 3. Basic equipment needed for the experimentation/laboratory
phase, and types of data that yielded the best success in my
classes.

                  Equipment                   Data

Pillbug Study     Mazes constructed of        Directionality of turn in
                  inert material (like Lego   a T-shaped maze Food
                  blocks), stop watch,        choice (carrot or potato
                  plastic spoon, small caps   extract offered)
                  to isolate isopods before
                  release, shoe boxes with    Time to complete maze
                  loose/fitting lids
                  prepared with plaster of    Time of day/date of
                  paris/carbonate mix for     trial
                  culture, and sponge in
                  dish for moisture           Trials run in
                                              triplicate (a)

Water Bear        Spring water, dissection    Total number of
Study             scope, pH meter, Pasteur    tardigrades at 24, 48,
                  pipettes, lichen            and 72 hours after
                  chemicals for testing,      immersion (a)
                  finger bowls, freezer,
                  thermometer, and graph      Number of different
                  paper to determine area     species at 24, 48, and 72
                  of lichen sample            hours (a) pH of tree bark

                                              Effect of temperature or
                                              moisture at collection
                                              versus number of
                                              tardigrades found

(a) During some terms my students removed animals at each
sampling, but in others they did not.
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