How-To-Do-It

Springtails in the Classroom

Collembola as Model Organisms for Inquiry-Based Laboratories
John C. Moore Bradley B. Tripp Rod T. Simpson David C. Coleman
Our objective is to advocate the use of springtails (Collembola) in the K12 classroom as a model invertebrate that can be easily reared and manipulated to demonstrate key concepts in biology. The National Science Education Standards (National Research Council 1996) and Benchmarks for Science Literacy (AAAS 1993) recommend activities that investigate and analyze questions, operate over several class periods, and use multiple process skills. Springtails are ideal organisms to use as teaching tools to meet these objectives (Usher & Stoneman 1977; Christiansen 1992). There are several advantages to using springtails: 1. Springtails can be easily isolated from soils, are small and require minimal space, food, and maintenance time. 2. Springtails are benign invertebrates that will not harm or pose any health problem to students or faculty. 3. There is a rich literature available on the identification, biology and ecology of springtails. 4. Springtail behaviors, response to stimuli, and interactions with each other and other organisms are easily observed with a simple hand lens. Springtails have been used as model arthropods in basic research in soil
John C. Moore is the Director of the Center for Precollegiate Studies and Outreach, and Moore and Bradley B. Tripp are in the Department of Biological Sciences at the University of Northern Colorado, Greeley, CO 80639. Moore and Rod T. Simpson are with the Natural Resource Ecology Laboratory at Colorado State University, Ft. Collins, CO 80523. David C. Coleman is with the Institute of Ecology at the University of Georgia, Athens, GA 30602-2360.

ecology, and have been used as test organisms for soil ecotoxicological work much as the cladoceran, Daphnia pulex, has been used for tests of water quality (Subaja & Snider 1981; Moore & de Ruiter 1993; van Straalen & Lkke 1997). One species, Folsomia candida (Willem), has earned the distinction of being the white rat or fruit fly of research in soil ecology and education (Usher & Stoneman 1977). The experiments outlined below were developed directly from basic research using F. candida.

Basic Biology & Phylogeny

Springtails are small (1 to 3 mm) sixlegged wingless arthropods that are commonly found in soils and decaying plant litter (Christiansen 1992). Springtails play an important role in the cycling of nutrients that are important to plant growth. Most species feed on soil, fungi, bacteria, algae and pollen, while a few feed on nematodes or roots. The phylogeny of springtails is currently being debated. Most authorities agree that they represent either a primitive form of, or are close relatives to, the insects. Like insects, the body of a springtail is divided into a distinct head that supports a pair of antennae, mandibles and maxillae; a three-segmented thorax that supports three pairs of walking legs; and an abdomen (Figures 1 and 2). Unlike insects, springtails possess a furcula that originates from the ventral surface of the abdomen. The furcula is normally held under the animal, however, if the animal were startled the furcula is r e l e a s e d , c a u s i n g t h e a n im al to spring forward to elude capture (hence the common name springtail). Species that live keep within the soil may not possess a furcula. Species that inhabit soil surfaces may be brightly and elaborately pig-

mented in different hues of purple or yellow, while those that live deep within the soil often lack pigment (appear white) and may lack eyes. Springtails possess a simple ametabolic life cycle in that only egg, juvenile and adult stages are present. Females lay small clusters of 10 to 20 spherical eggs in protected crevices within the soil or litter. Development time varies by species and with temperature and moisture, however a typical gestation period at room temperature is approximately two weeks. The juveniles appear as small (nonsexual) versions of the adult with muted pigmentation if the species are pigmented. Springtails grow and molt (shed their exoskeleton) in typical arthropod fashion. The juvenile stage involves three successive instars lasting 7 to 10 days separated by a molt. Adults continue to molt throughout their lives and can live for up to a year (Snider 1973). Springtails are dioecious, having either male or female reproductive organs. However, for many species the males are extremely rare. The species that is highlighted in this paper, Folsomia candida (Willem), is parthenogenetic (Goto 1960). The ovum develops (without fusion with a sperm) into diploid copies or clones of the female. Males of F. candida have been recorded in wild populations.

Collecting & Rearing Springtails

Springtails are readily collected from soils and leaf litter using a modification of the Tullgren funnel (Figure 3). A sample of soil or litter is collected using a small shovel or spade and placed in a Ziploc storage bag. The bags should be placed in a dark cooler out of direct sunlight as the light passing through the bag will heat and kill the animals. In the laboratory, the

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be transferred from one jar to another by tapping them from one source onto a piece of wax paper and then funneling them into the other source. Secure the lid tightly and store the jars at or below room temperature in a cool place out of direct sunlight and away from a heat source. Check the culture weekly (we have let cultures go unchecked for months) to add water and yeast when needed, and to remove any fungus that may have grown on the yeast. Eggs and juveniles should appear within two weeks after the culture has been initiated. We recommend that the animals be transferred to new jars every 1 to 2 years, and that jars be discarded if cultures die out completely. The special dietary requirements of many species are unknown. Rearing certain species can be challenging and, in and of itself, offers an excellent opportunity for inquiry.

Examples of Laboratory Exercises

We offer the following two laboratory vignettes that were conducted by a sixth- and eighth-grader for their science fair projects: 1) The effects of temperature on the life history of F. candida, and 2) The effects of 2,4-D and Banvel on the life history of F. candida. Both studies were completed within 7 weeks, but could easily fit within a 3 to 6 week block for K12 schedules. We recommend that the first week be devoted to discussion of the problem, experimental design, and preparing the culture jars. A class period should be devoted to covering the methods of data collection, formulating data sheets, and developing a schedule for data collection.

Figure 1. Schematic diagrams of the elongated (A) and globular (B) body forms. A) The dominant morphological characteristics of a springtail: 1) antenna, 2) postantennal organ (P.A.O.), 3) eyes, 4) leg, 5) collophore and 6) furcula.
sample is gently loosened and wrapped in cotton cheesecloth (not polyester, as animals tend to get caught in the fibers) and placed over a piece of 14 inch hardware cloth that is placed in the mouth of a large funnel. A low wattage ( 15 watts) incandescent light source is placed 2 to 4 inches over the sample (5 days) and a collection jar containing water for live collections or 70% ethanol (simple rubbing alcohol will suffice) for preserved collections is placed under the spout. As the heat from the light warms and dries the surface of the sample, the springtails (and other small arthropods) move down through the sample and eventually make their way out the bottom, down the funnel, and into the collecting jar. An aluminum foil jacket wrapped around the light and mouth of the funnel will focus the heat toward the surface of the sample and prevent animals from escaping from the sides. Live animals can be collected from the water surface of the collection jar their weight is easily supported by surface tension. They can then be transferred to rearing jars with the moistened bristles of a small paintbrush. Small plastic or glass jars (e.g. medicine vials, baby food jars, or pint-sized jelly jars) lined with a 9:5 mixture of cured plaster of Paris and powdered charcoal (Fisher Scientific Catalog #C-170) make an ideal substrate to rear springtails (Snider et al. 1969). The plaster and charcoal should be mixed to a creamy, peanut butter-like consistency. A dollop of the mixture is placed in the jar and is spread over the bottom by gently tapping the bottom of the jar on a tabletop. After the mixture has cured, add enough water to completely wet the plaster but not so much that standing water is visible. Place the springtails in the jar and add a few granules of bakers yeast as a food source. If jars are checked weekly, no air holes are needed. Springtails can

Vignette 1. Effects of Temperature on the Life Cycle of Folsomia candida

The student was presented with two observations generated from a survey of the sediments of Wind Cave and Jewel Cave, South Dakota. The dominant species in both caverns is F. candida (Willem). Wind Cave supported higher incidences and densities of springtails than Jewel Cave. One factor that could explain the differences in occurrence and density between the caverns is their mean annual temperatures: 13 C in Wind Cave and 9 C in Jewel Cave. The temperatures of both caves were well within the tolerance range of F. candida and other
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Figure 2. Photographs of slide mounts of the dominant families of springtails possessing the globular and elongated (A-E) body forms. A) Family: Isotomidae (Folsomia candida) (100x), B) Family: Sminthuridae (100x magnification), C) Family: Onychiuridae (100x), D) Family: Hypogastruridae (100x), E) Family: Entomobryidae (40x).

species, but below their optimum (Snider 1973). The objective of this study was to determine whether the differences in temperature could explain the lower incidence number and densities of F. candida. Given that springtails are ectotherms and the observations from the survey of the caves, the student formulated the hypothesis that the differences were due to declines in growth and reproduction at the lower temperatures. From this hypothesis, the student predicted that F. candida

reared at the lower temperatures associated with Jewel Cave would exhibit less growth and reproduction than those reared at higher temperatures found in Wind Cave. The variables compared were mortality, the number of molts, the duration of an instar, eggs produced, the development time of the eggs, and the number of juveniles produced. Materials & Methods Twenty culture vials were established using the materials and tech-

niques described above. F. candida obtained from a neutral location were used in the experiments. Three adult F. candida ( 1 mm) were placed in each vial with yeast as a food source and water. Ten cultures were randomly assigned to one of two incubators set at 10 C and 15 C, meant to represent Jewel Cave and Wind Cave, respectively. We chose slightly higher temperatures to represent each cave to account for the upper end of the variation in temperature within the caves and for convenience. Each vial

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Mortality data were analyzed using categorical analysis. All other variables were analyzed using analysis of variance. Prior to analysis of variance, response variables were transformed as ln (x 1). Unless otherwise stated, all significant results were reported at the p 0.05 level. Results A summary of mortality, molts, egg production, and juveniles produced is presented in Table 1. Cultures maintained at 10 C suffered higher mortality (26.3%) than those maintained at 15 C (9.3%). Within the first week, both sets of cultures lost a single individual. This was not entirely unexpected, given the handling and the likelihood of an individual dying of natural causes. By Day 12, 2 additional individuals expired in the 10 C cultures. At the end of the experiment, 8 of the 30 animals had died at 10 C, with 1 culture completely depleted, while only 2 had expired at 15 C. Animals reared at 15 C molted significantly more frequently, laid significantly more eggs, and produced significantly more juveniles than those reared at 10 C during the 40-day period (Figure 4). On Day 40, egg production in the cultures did not differ significantly. The differences in mortality could influence the interpretations of the results for the number of molts, eggs and juveniles. We present these results and will address how mortality may have affected them below. The cumulative molt data indicate that the instar duration of F. candida reared at 15 C and 10 C was about 15 and 30 days, respectively. This could be deduced by assessing the point at which the number of molts Table 1. Summary statistics of mortality, molts, eggs and juveniles for cultures of Folsomia candida reared at 10 C and 15 C for 40 days. Temperature 10 C Number of Jars Animals per Jar Mortality (%) Molts Eggs Juveniles 10 3 26.7 26.0 177.0 1.0 15 C 10 3 9.3 49.0 378.0 148.0

Figure 3. Photograph of a modified Tullgren funnel fashioned from a standard ring stand. A) light source, B) funnel with hardware cloth supporting a soil sample wrapped in cotton cheesecloth, C) collection jar with 70% ethanol for preserved collections. For live collections, substitute the ethanol with water or a jar containing the 9:5 charcoal to plaster mixture described in the text. was inspected for the number of eggs, molts, juveniles and live collembola every 3 to 5 days for 40 days. A simple dissecting scope was used to count eggs and molts. Juveniles and adults could be seen with the naked eye, but a hand lens was used in this experiment. The student carefully inspected cracks or holes in the surface of the substrate to find small clusters of eggs.

a* a a a

b b b b

*Different letters differ at the p 0.05 level. Total number of eggs as the number of eggs plus juveniles on Day 40. Total juveniles estimated as the sum of the number of juveniles per jar on Day 40.
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per culture averaged multiples of 3 molts. When comparing 2 treatments, the more conservative estimate of molting frequency would assume that all the mortality occurred at the onset, and hence adjust for the deaths. Given the number of survivors and the total number of molts over 40 days, the molting frequency was 1.18 molts per individual at 10 C, and 1.75 molts per individual at 15 C. Given these estimates, the instar duration for 10 C and 15 C is 33.9 and 22.8 days, respectively. The molting frequency appears to have declined to a steady state by Day 32 for cultures reared at both temperatures, as there was no significant gain in the cumulative number of molts from Days 32 to 40. Egg production was delayed in the cultures maintained at 10 C compared to those maintained at 15 C. Of the 10 cultures reared at each temperature, only 1 reared at 15 C failed to produce eggs, compared to 5 for 10 C. Egg production peaked on Day 23 with 59.9 38.5 eggs per jar for the 15 C cultures, while those reared at 10 C reached their highest numbers at 17.6 21 eggs per jar on Day 40. When we accounted for mortality as above, egg production at 10 C and 15 C was 8.0 and 13.5 eggs per individual, respectively. On Day 5, a single juvenile was recorded in a culture reared at 10 C. This individual was observed on all days except Days 15 and 23. Only one additional juvenile was recorded in the 10 C cultures (on Day 40). It was concluded that the first was either inadvertently added to the culture along with the 3 adults at the onset of the experiment, or was added as a single egg and hatched soon after. Seven of the 10 cultures maintained at 15 C produced young. For these cultures, the first juveniles appeared on Day 23 and increased thereafter to 12.9 12 juveniles per jar. When the mortality was considered, juvenile production at 10 C and 15 C was 0.05 and 5.29 juveniles per individual, respectively. Discussion The student had hypothesized that the higher densities and frequency of occurrences of F. candida in Wind Cave compared to Jewel Cave were due to declines in growth and reproduction of the animals at the lower temperatures found in Jewel Cave. The results presented above support the hypothesis. Figure 4. Life history measurements of Folsomia candida reared at 10 C and 15 C over the course of the 40-day experiment. Each graph depicts the mean per jar standard error of A) cumulative number of molts, B) number of eggs, C) the number of juveniles.

Vignette 2. Effects of Herbicides on the Life History of Folsomia candida

A student from a rural community expressed an interest in how pesticides

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might affect nontarget organisms. Moreover, the student had learned in class that many organisms developed resistances to pesticides. Assessing nontarget effects would employ methods similar to those presented in the first vignette, where treatments would include exposure to the pesticide and unexposed controls, and the response variables would include mortality, molts, eggs and juveniles. Assessing resistance to pesticides was another matter, as resistance, if it were to occur, would take several generations. The alternative would be to identify a population that has been spared the exposure from pesticides to one that has had routine exposure. It was fortuitous that at the time the student was making this inquiry, our research group had isolated F. candida from the sediments of Wind Cave and Jewel Cave in South Dakota. We assumed that these animals had not been exposed to pesticides. We decided to compare the responses of F. candida from the caves (South Dakota) to those that were isolated from an agricultural field (Georgia). The student formulated two null hypotheses. The first hypothesis stated that the herbicides would have no effect on the life histories of F. candida. The student predicted that F. candida exposed to pesticides would experience the same mortality, growth and reproduction as untreated controls. The second hypothesis stated that the responses in life history of F. candida isolated from South Dakota and Georgia following the application of the herbicides would not differ. Materials & Methods Twenty-four culture jars (standard medicine vials) were prepared using the materials and techniques described

Figure 6. Head region and examples of setae. a) Examples of the postantennal organ (P.A.O.) and b) Pseudocelli. Examples of uniciliate (c and d) and multiciliate (e) setea and (f) scales. above. Stock cultures of F. candida isolated from the sediments of Wind Cave and from an agricultural soil in Georgia were maintained for at least one year prior to the experiment at room temperature. Half of the 24 jars each received 10 F. candida from South Dakota, and the remaining half each received 10 F. candida from Georgia. For each of 2 groups of 12 jars, 4 were randomly designated untreated controls, 4 as Banvel treatments, and 4 as 2,4-D treatments. Jars designated as controls were treated with 0.1 ml distilled water, while those treated with the herbicide received a 0.1 ml solution mixed to field strength as recommended by the manufacturer. All jars were maintained at room temperature. Each jar was inspected with a hand lens daily for 2 weeks for the number of eggs, molts, juveniles, and living collembola. Mortality data were analyzed using categorical analysis. All other variables were analyzed using analysis of variance. Prior to analysis of variance, response variables were transformed as ln (x 1). Unless otherwise stated, all significant results were reported at the p 0.05 level. Results A summary of mortality, molts, egg production, and juveniles produced are presented in Table 2. Animals suffered higher mortality in the Banvel (23.8%) and 2,4-D (42.5%) treated jars than the control (10%) jars. Differences between cultures were also apparent, with slightly higher mortality found in the treated jars from Georgia than those from South Dakota (Table 2). The cultures from Georgia produced significantly more eggs (3.2 23.5 per jar) than those from South Dakota (0.6 14.5 per jar). The high variance associated with these means suggests that this is a bit misleading, as there was a significant treatment by culture interaction (p 0.10). Egg production in the untreated control cultures did not differ significantly. However, cultures from South Dakota that were treated with either herbicide failed to produce any eggs, while the highest production was found in the treated jars from Georgia (Table 2). Juvenile production mirrored egg production, but given the low numbers and high variability, the analysis of variance indicated that the culture and treatment means did not differ significantly. Animals from Georgia molted more frequently than those from South Dakota. Although the cultures differed in the number of molts, animals from both sources reared in control jars were
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Figure 5. Prothorax without (A) and with (B) dorsal setae. This character is used to distinguish families of the elongated body forms. The Hypogastruridae (Figure 2D), Onychiuridae (Figure 2C) and Poduridae possess dorsal setae on the prothorax, while the Isotomidae (Figure 2A) and Entomobryidae (Figure 2E) do not.

Figure 7. Dorsal view of a furcula with spines. (A) 1) manubrium, 2) dentes, 3) mucro, 4) spines. Many species do not possess spines, but do possess simple setae (Figure 6 c, d). Lateral view of furcula with crenulate dentes (B) and smooth dentes (C).

Table 2. Summary statistics of mortality, molts, eggs and juveniles per jar for control and herbicide-treated jars from Georgia (GA) and South Dakota (SD) after 14 days. Control GA Number of Jars (n) Animals per Jar Mortality (%) Molts Eggs Juveniles 4 10 10.0 14.9 1.7 1.2 SD 4 10 10.0 3.2 1.4 1.2 GA 4 10 30.0 3.6 4.1 1.5 Banvel SD 4 10 17.5 2.4 0 0 GA 4 10 50.0 5.7 3.8 1.2 2,4-D SD 4 10 35.0 1.7 0 0

have been maintained for 10 and 5 years, respectively. We have used variations of the studies discussed above with high school students and in undergraduate ecology labs. The labs spanned 3 to 6 weeks, and required 1 to 3 hours of actual hands-on attention by the students. Once completed, the labs lend themselves to written reports, science fair posters, and talks. These activities can be structured to allow students to investigate questions, analyze data, and improve science process skills. Instructors can provide students with as little or as much information as they deem appropriate. It is important to reinforce a few of the basic science process skills. Initial conditions should be identical for all treatments. Treatments should be randomly assigned. Control groups are an essential part of a good experiment. Data to be collected should be identified prior to starting the experiment. Springtails are just one of a myriad of soil organisms. Mites, nematodes and protozoans are all common soil organisms. Some time ago the Biological Sciences Curriculum Study (BSCS) produced a laboratory block titled Life in the Soil as a 6-week laboratory unit (Pramer 1965). While slightly dated, it is still an excellent introduction to soil as a living organism.

Acknowledgments
We wish to thank Evan Moore and Maurissa Moore for their work with springtails and presentations at local and regional science fairs. Their efforts provided insights into the feasibility of working with the organisms in school and home settings. We also thank Ryan Fox and Renee Jesser for their help in maintaining the springtail cultures and monitoring the experiments. This work was supported in part by grants from The National Science Foundation (DEB-9257710) and U.S. National Park Service.

a* a a a

a b a a

b b a a

ab bc b a

b b a a

b c b a

*Different letters differ at the p 0.05 or p 0.10 as stated in the text. twice as likely to molt than those reared in jars treated with the herbicides (p 0.10). Discussion The student rejected both hypotheses. The herbicides had significant effects on the life histories of F. candida, regardless of where the animals came from. While significant differences in mortality and life history between the cultures were found, interpreting them was another matter. The student had expected the culture from the agricultural fields from Georgia to show lower mortality than those collected from the caves in South Dakota, when in fact the opposite happened. However, the populations from Georgia demonstrated greater resistance through molts and reproduction.

Concluding Remarks
We have used springtails to teach basic principles of biology and ecology to students at the middle school through graduate school levels. From simple investigations of life histories to more complex experiments involving predator-prey interactions, springtails offer an affordable model that fits within the time, space and budgetary constraints of many schools. Once established, cultures of F. candida are extremely robust. For example, the cultures from Georgia and South Dakota

References
American Association for the Advancement of Science (1993). Benchmarks for Scientific Literacy. New York: Oxford University Press. Christiansen, K. (1992). Springtails. The Kansas School Naturalists, 39, 316. Christiansen, K. & Bellinger, P. (1980). The Collembola of North America: North of The Rio Grande. Grinnell, IA: Grinnell College.

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Goto, H.E. (1960). Faculative parthenogenesis in Collembola (Insecta). Nature, 188, 958959. Moore, J.C. & de Ruiter, P.C. (1993). Assessment of disturbance on soil ecosystems. Journal of Veterinary Parasitology, 48, 7585. Moore, J.C., Ingham, E.R. & Coleman, D.C. (1987). Inter- and intraspecific feeding selectivity of Folsomia candida (Willem) (Collembola, Isotomidae) on fungi. Biology and Fertility of Soil, 4, 17. National Research Council (1996). National Science Education Standards. Washington, DC: National Academy Press.

Pramer, D. (1965). Life in the Soil. Biological Sciences Curriculum Study. Lexington, MA: D.C. Heath and Company. Snider, R.J., Shaddy, J.H. & Butcher, J.W. (1969). Some laboratory techniques for rearing soil arthopods. Michigan Entomologist, 1, 357362. Snider, R.J. (1967). An annotated list of the Collembola (Springtails) of Michigan. The Michigan Entomologist, 1, 179234. Snider, R.M. (1973). Laboratory observations on the biology of Folsomia candida (Willem) (Collembola: Isotomidae). Revue Ecologie Biologie du Sol, 10, 103124.

Subaja, J. & Snider, R.J. (1981). The side effects of the herbicides atrazine and paraquat upon Folsomia candida and Tullbergia granulata (Insecta: Collembola). Pedobiologia, 22, 111152. Usher, M.B. & Stoneman, C.F. (1977). Folsomia candida: An ideal organism for population studies in the laboratory. Journal of Biological Education, 11, 8390. Van Straalen, N.M. & Lkke, H. (1997). Ecological approaches in soil ecotoxicology. In N.M. Van Straalen & H. Lkke (Eds.), Ecological Risk Assessment of Contaminants in Soil (pp. 321). London: Chapman Hall.

Appendix
A Key to the Families of Springtails (Collembola)
[Adapted from Snider (1967) and Christiansen & Bellinger (1980)]
1) Body elongate, furcula present or absent (Suborder Arthropleona; Figures 1A, 2BE) ......................... 2 Body globular, furcula always present (Suborder Symphypleona; Figures 1B, 2A ................................ 10 2) Prothorax distinct when viewed dorsally and with dorsal setae (Figure 5B) ........................................... 3 Prothorax reduced, hidden from dorsal view and without setae (Figure 5A) .......................................... 6 3) Dentes more than 3 times as long as manubrium .............................................................................Poduridae Dentes absent or less than 3 times as long as manubrium ........................................................................... 4 4) Pseudocelli present, at least on antennal base, or on dorsum of fifth abdominal segment (Figure 5A) ............................................................................................... Onychiuridae Pseudocelli absent ........................................................................................................................Hypogastruridae 5) Third antennal segment much longer than fourth segment, both segments subsegmented; mucro hairy .................................................... Entomobryidae (subfamily Tomocerinae) Third and fourth antennal segments simple; mucro with at most 1 to 2 setae ........................................ 6 6) Dens with dental spines, mucro may be longer than dentes (Figure 6a) .......................... Entomobryidae (subfamily Oncopodurinae) Dens without spines, or if present simple; mucro much shorter than dentes .......................................... 7 7) P.A.O. present, or if absent setae unilaterally ciliate (Figures 6b,c,d) ........................................ Isotomidae P.A.O. absent, some setae multilaterally ciliate (Figures 6e,f) ...................................................................... 8 8) Dens crenulate (Figure 7B) ....................................................... Entomobryidae (subfamily Entomobryinae) Dens smooth (Figure 7C) ..................................................................................................................................... 9 9) Eyes and pigment absent ............................................................ Entomobryidae (subfamily Cyphoderinae) Eyes and pigment present ............................................................ Entomobryidae (subfamily Paronellinae) 10) Antennae shorter than head; eyes absent ............................................................................................ Neelidae Antennae usually longer than head; eyes present .................................................................... Sminthuridae

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