How-To-Do-It

As the Worml Turns Locomotionin a FreshwaterOligochaete Wlorm Charles Drewes Kacia Cain

Worms are generally perceived as behavior is called a central pattern Materials neither very manageable nor talented generator (Young 1989). Central pat- with respect to their locomotor abili- tern generatorsfor locomotionin anne- * Lumbriculusvariegatus (use several Downloaded from http://online.ucpress.edu/abt/article-pdf/61/6/438/49035/4450725.pdf by guest on 25 September 2021 ties. However, locomotion in black- lids and arthropodsare usually located blackworms/group).Sources are: worms may be an exception and, con- in the ventral nerve cord. Motor neu- (1) www.novalek.com/korgdel.htm sequently,we hope this articlechanges rons in the ventral nerve cord are (2) www.holidayjunction.com/aro/ such "wormy" perceptions. responsible for conveying impulses (3) www.carolina.com Blackworms, Lumbriculusvariegatus from the central pattern generator to (4) tropicalfish and pet stores. (Phylum Annelida, Class ), the specific muscles in the body that For additionalbiology background are common in wetlands of North produce locomotor movements. about Lumbriculus, see Drewes America. Unlike tubifex worms (dis- Many forms of rhythmiclocomotion (1996a,b) and Lesiuk & Drewes tant relatives that occupy tunnels in depend on the coordinated actions of (1999). muddy sediments), Lumbriculusfreely well-designed appendages. Other * Disposable petri dishes (150 x 20 crawls on submerged and decaying forms of locomotion require no mm): one-half dish/group vegetation, such as decomposing appendages,such as peristalticcrawling * Disposable petri dishes (60 x 15 leaves, logs and cattails.When touched in many terrestrial, freshwater and mm): one-half dish/group or threatened,it uses a variety of loco- marine worms; or undulatory * Disposable petri dishes (100 x 15 motor responses to protect itself or swimmingin certain aquatic , mm): Each student group will use move to safety. One of these responses, some nematodes, and some snakes one dish, which should be half- helical swimming, is a fascinatingand (Trueman 1975; Drewes & Fourtner unusual form of locomotion. 1993; Drewes 1999). filled with springwater and contain several worms. An early version of this exercisewas The experiments here focus on field tested by high school biology crawling and swimming behavior in * Filter paper disks (12.5-cmdiame- teachers at a 1996 Summer Institute Lumbriculusvariegatus, a freshwateroli- ter): one disk/group in Neurobiology (Woodrow Wilson gochaete worm. Many questions arise * Plastic dropping pipet (6"plain, or National Leadership Program for about the control and biomechanics eye dropper):one/group Teachers;Princeton, NJ). Also, we pre- of this worm's locomotion.Answering * Spring water (about 200 ml/ sented the material as a "hands-on" these will require your students to group).Most commercialbrands of workshop at the 1996 NABT meeting make close observations, repeated spring water (forexample, Evian", (Charlotte,NC). measures, and keen insights. NayaT, Poland Springs") are very "worm friendly." Have several large containers of spring water Background available. [NOTE:Well-aged and General Suggestions dechlorinatedtap water may be an Locomotionis crucial to animal sur- Lumbriculus,like many freshwater acceptable alternative to spring vival because it enables movement water in many cities. To test toward food or a mate, or away from invertebrates,may die if exposed to small amounts of chlorine, formalin, whether aged water is safe for unfavorable habitats or predators. worms, place two to three worms Often, locomotion involves rhythmic dishwasher soap residues, and many other lab chemicals. Make sure glass- in a small, covered containerof the movements, such as walking, running, water overnight and confirm that and 1968; ware, plastic ware, pipets, and other swimming flying (Gray worms are alive the next day.] Alexander 1982). The specific network items used in these experiments are * 51/2"x 7/8" or neurons in an animal's nervous free of these chemicals. If in doubt, Swim chamber(prefered: system that controls such rhythmic thoroughly rinse items with dechlori- disposable plastic weighing boats, nated tap water or spring water. For containing 100 to 120 ml of spring cleanup, wash and rinse items in tap water). For container strength, we CharlesDrewes is Professor of Zoology water using no soap. Items should be recommend stacking three weigh- and Geneticsat IowaState University, completely dry before reuse. ing boats together. Alternative Ames, IA5001 1; e-mail: cdrewes@ in small swim chambersshould be about 10 iastate.edu. Kacia Cain is Chairper- Students should work son of Science at East HighSchool, groups (two to three students/ group). to 20-cm diameter,2 to 4-cm deep, Des Moines,IA 50316. ExperimentsI and II require about 20 and have a smooth, light-colored to 30 minutes each to complete. bottom.

438 THEAMERICAN BIOLOGY TEACHER, VOLUME 61, NO. 6, JUNE1999 * Plain wooden applicator stick, or ExperimentI: Forward& of the marked dish (Figure 2B). With toothpick (two/ group) gentle handling, patience and practice * Rubber bands (narrow, size #19; RearwardCrawling you will get the worm to crawl around four/ group) A) Prior to class, use a permanent and around this track. If, by chance, * A few human hairs (straight, markingpen and flexible ruler to care- the worm roams far away from the coarse, about 1"-long;two/group) fully make a series of half-centimeter track,or begins crawlingoff the paper, * Maskingor label tape (severalrolls) marks around the outside edge of a don't panic or force the worm back * Coins (about 2 to 3/group; not 6-cm plastic petri dish. Number whole into position! Simply, tilt the crawling for betting) centimeter marks consecutively so platform slightly, and use one or two * Whiteunlined paper (as light back- numbers can be read when the open pipets full of spring water to flush the ground under worm containers) side of the dish is facing down (see Fig- worm off the paper and into the runoff * Metricrulers (flexible vinyl, 15-cm ure 2A). water at the edge of the dish. Then, length; one / group) B) Place a 150-mmpetri dish on the suck up the worm, deposit it again next to the track. Tilt the dish and * Stop watch, wristwatch, or room counter top with its open side facing up; this dish is the crawling platform. remove excess water. Then, add back clock (must show seconds) 4 ml of spring water, as described * Permanentmarking pens (narrow Next, center a 12.5-cmfilter paper disk in the dish and saturateit completely above in Step D. Worms may be tip; one per group) unwilling to stretch out and crawl if * Scissors (to cut rubber bands and with spring water. a worm as the filter paper is too dry. tape; several per class) C) Assemble "racetrack" shown in Figure 2A. The coins, taped G) Study forward and rearward Downloaded from http://online.ucpress.edu/abt/article-pdf/61/6/438/49035/4450725.pdf by guest on 25 September 2021 * 10-ml graduated cyclinder (one/ to the centerof the inverted inner petri crawlingmovements around the track. group) dish, provide an elevated surface that To evoke forward crawling,very gently is higher than the edge of the outer stroke tail segments with the rubber tip. To evoke rearwardcrawling, gently Optional Materials dish. The rubber bands, crisscrossed over the coins, provide downward stroke head segments. During crawl- pressure to firmly hold the open face ing, note areas where the worm's body * Stereo dissecting microscope of the inner dish against the paper. appearsthicker and darker(Figure 2B). (one/group) This prevents worms from crawling In these areas, segments have short- * Pipe cleaners or solid-conductor, under the edge of the inner dish, which ened, due to longitudinal muscle con- insulated wire for models of swim- is undesirable. traction. Note also that these areas ming worms D) Use a pipet to transfer a worm move, as a wave, along the body in * Wooden dowels (/4, 5/16, and 3/8") for from the 10-cm storage dish onto the a direction opposite the direction of models of crawling worms filter paper on the crawling platform, locomotion. * Video camcorder close to the inner dish. Tilt the crawl- * Tripod ing platform at a 450 angle and use [Note:Upon request, authors will provide and * Adjustablelighting the pipet to remove the accumulated neurobiologybackground information water at the edge of the dish. Then, answers to the following two question add back 4 ml of spring water to the sets]. paper, thus insuring that the paper Assembling WormWidgets will be sufficientlysuper-saturated for & Racetrack good crawling performance by the Questions About Crawling worm. Cl) Give possible reasons why the Touch stimuli readily evoke locomo- E) Now, make sure you can distin- worm usually crawls in a circularpath tion in Lumbriculus.However, since guish between the worm's head and around the dish rather than in a worms are easily damaged by sharp, tail. The head end is darker, thicker, straight line. rigid objects, small probes (widgets) and generally more active. The tail is C2) In what direction do peristaltic will be used to harmlessly touch light-colored, thin, and less active. waves of contractionmove along the worms. Carefully assemble each wid- F) Use the tip of the straightrubber worm's body as it crawls forward?In get by taping pieces of rubber band widget to verygently touch the worm's a head-to-taildirection? Or tail-to-head and hair to applicatorsticks, as shown head or tail end until it begins crawling direction?In what direction do waves in Figure 1. forward or backwards along the edge move during rearward crawling? C3) Since peristaltic waves are rhythmicand repetitive,try to measure the time (in seconds) between two straight looped straight looped successive waves as these move hair hair rubber rubber throughone specificbody region, such as 1 cm posterior to the head end. -f112in Then, calculate the wave frequency (expressed in waves/minute). Repeat - tape these measuresat least three times each for forward and rearwardcrawling. -- applicator C4) Watchthe worm crawl for three to four minutes without touching it. stick Does forward or rearward crawling ever start spontaneously? C5) Test whether a strong stimulus (straightrubber widget) is more effec- Figure 1. Widgets for touching worms. tive than a weak stimulus (straighthair

LOCOMOTIONIN A FRESHWATEROLIGOCHAETE WORM 439 three times to obtain a range of veloc- ity values, expressed in units of cm/s and meters/minute. Measure the worm's body length. When crawling fast, how long does it take the worm to crawl a distance equal to its body length? Or the length of a 2-m log? C8) Remember that each body seg- ment of the worm is a fluid-filled, non-compressiblecompartment whose

} ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... !. . . volume is essentially constant. Then, describe the changes in segment shape that occur as peristaltic waves move along the worm and how these changes relate to contractionby circu- lar and longitudinal muscle layers (see Figure 3). Also, describe the pushing and pulling forces on neighboringseg- ments that occur as peristaltic waves move. Downloaded from http://online.ucpress.edu/abt/article-pdf/61/6/438/49035/4450725.pdf by guest on 25 September 2021 ; _, 5.>. 2 ,e, ...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..c,\h>...... C9) Use a stereo microscope to esti- .~~~~~~~~~~~~~~~~~~~...... _o?.@!,o mate the number of segments . . to l t14 41li l ulull_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.tLA1<...... involved, at any instant of time, in _~~~~~~~~~~~~~~~~~~~...... one wave of longitudinal muscle con- _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.../ ..... traction (that is, where body segments appear thickened). Estimate the num- ...... ber of segments in a circular muscle wave, where segments appear thinner. C10-BONUS).If circular muscle in

_ ; ...... _._. . .. a segment contracts so that segment diameter is reduced by one-half,how much does the segment length * X. . i \h..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....;...... | ...... increase?Show calculations.Note that cylinder volume = w - radius2 *length. Assume segment volume is constant. t : :?: . . .,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... :....'?.j..-isl- ^ . .:' . ' .': ; ...... i,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...:i..;''.]l,':''''.''',.-,e,...... Experiment 11:Undulatory 1t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...t'=..'..'.. Swimming & Reversal A) Transfera new worm to a swim chamberfilled with 1 to 2 cm of spring water. If needed, place white paper under the chamberfor a white viewing ...... : :; . ' ...... , . :: s i . ^ } ' ' ' ' ' . X ?~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... n background. Identify head and tail ends. B) Use the rubber loop widget to gently coax the worm to the center of L.C . : .. the chamber. Then, with the widget positioned at an angle of about 450 (see Figure 4A), gently and quickly Figure 2. Worm "racetrack." a) Criss-crossed rubber bands hold the inverted dish touch the loop down on the tail for (60-mm D petri dish, labeled ID) against the water-saturated filter paper placed in a brief instant. If done properly, this the outer dish (150-mm D petri dish, labeled OD). b) Close-up view of a worm stimulus does not injure or trap the crawling around the marked, inner dish. Note the wave of longitudinal contraction worm. Instead, the worm responds by (LC) where body segments are thickened. H, head end; T, tail end. producing a series of brief and very rapid swimming movements that pro- pel it forward and away from the widget) in evoking forward crawling. tail to head.) The result is a map of the stimulus. Repeat this for rearward crawling. touchsensory field for forwardcrawling. Although movement details are too C6) Make a sketch of the worm; Obtain a similar map of the touch rapid to see, the worm actually swims label head and tail ends. On the sketch, sensory field for rearward crawling. bending its body into the shape of show all locations along the body C7) Obtain estimates of forward a helical twist (Drewes & Brinkhurst where touching usually evokes for- crawling velocity. Do this by noting 1990). Bending movements rapidly ward crawling. Indicate where it fails the distance the head end moves dur- move, as a wave, from the head to to do so. (Note: Try touching at least ing a 10-second interval of crawling. tail. Rearwardprogress of each helical six to eight different locations along This may require continual or repeated wave acts against the water and pro- the body, moving progressively from touching of the tail. Repeat at least vides thrust that propels the worm

440 THEAMERICAN BIOLOGY TEACHER, VOLUME 61, NO. 6, JUNE1999 forward. Curiously, successive waves alternate as clockwise and counter- 1 2 3 2 34 clockwise helical twists (Figure 5). C) Repeat Step B, this time briefly touching the loop on segments near 4Q11Lz1 the head (Figure 4B). Now, the worm's response is a "reversal" behavior in 2 ~~~~~~~~~4(- which it quickly curls and then uncurls its body, causing a near 1800 reversal of its body. In effect, head and tail reverse positions without a major shift in the body's center of mass. This is a nifty biomechanical feat, done without much benefit of traction with the Figure 3. Diagram of hands-on model of crawling worm. The model has four types smooth bottomed dish. of interchangeable pieces made from wooden dowels: 1) head-piece 1/4"D; 2) mid-piece undergoing longitudinal muscle contraction wave 3/8" D; (3) mid-piece undergoing circular muscle contraction wave 1/4"D; and (4) tail piece-1/4" D. Questions About Segmentation patterns in mid-pieces are shown to approximate scale and are made by wrapping dowels with pre-lined adhesive labels and clear tape. Dots in #2 mid- Swimming & Reversal piece indicate protruding chaetae that increase traction. Segmentation patterns in Downloaded from http://online.ucpress.edu/abt/article-pdf/61/6/438/49035/4450725.pdf by guest on 25 September 2021 head and tail pieces are not shown. Pieces are linked by metal wire inserted and S1) Does swimming or reversal ever glued into matching holes. occur spontaneously? S2) Use the hair loop widget (weak stimulus) and rubber loop widget (strong stimulus) to determine whether weak stimuli are as effective as strong ones in evoking swimming and reversal. S3) Sketch the worm. Label head and tail ends. On the sketch show all locations along the body where touch evokes swimming and all locations where it fails to do so. To do this, touch at least six to eight different locations along the body, moving pro- ...... gressively from tail to head. Your data :~~~~~~~~~~~~~:X~~~~~~~~~~~- .~ ~~~~~~~~ result in a map of the touch sensory --,..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... field for swimming. Why do you think this field does not include anterior segments? Obtain a similar map of the touch sensory field for reversal. Figure 4. Worms in the swim chamber. a) The rubber loop widget is used to Compare and contrast these maps to stimulate the worm's tail end (T) and evoke swimming. b) The loop is used to the ones obtained for forward and stimulate the head end (H) and evoke reversal. rearward crawling (Question C6). S4) Is there any apparent stimulus that stops swimming? S5) Estimate (in seconds) how long Optional: Video Analysis Optional: Locomotion by a swim episode lasts and (in centime- of Locomotion Body Fragments ters) the total distance the worm swims. Speculate how such short-dis- Obtain closeup, video recordings of Lumbriculushas a remarkable ability tance swimming could help avoid swimming and reversal using a video to reproduce asexually by self-frag- predation. camcorder with variable shutter speed mentation. Even very short body frag- S6) Estimate the amount of time (in and closeup (macro focus) features. ments survive and rapidly develop seconds) it takes for a reversal and Shutter speeds of 1/500th or 1/1000th into whole worms by regenerating describe changes in head and tail posi- second give clear, freeze-frame images missing head and/ or tail segments tion before and after several reversals. of the worm's swimming movements. (Drewes & Fourtner 1990). As options, S7) Test whether swimming res- Replay taped episodes using a VCR we suggest repeating Experiments I ponses eventually fail to occur (habitu- with single-frame advance. Try to dis- and II using anterior and posterior ate) when stimulation is repeated. To cern clockwise or counter-clockwise worm halves or smaller pieces. See do this, repeatedly touch the worm's helical twisting of the body and calcu- Drewes (1996b) for methods in obtain- tail with the rubber loop at intervals late the frequency of helical waves ing fragments and studying regenera- of 30 seconds. How long does it during a swim episode. Recall that the tion in Lumbriculus. take before the worm's swimming time between each video frame is 1/ Amazingly, response patterns in responses become less vigorous or fail? 30th second. Place a transparency sheet body fragments rapidly switch right About how long does it take to fully over the monitor screen and trace out- after cutting, so that each body frag- recover responsiveness? (1 minute? lines of swim movements in succes- ment can crawl, swim and reverse on 5 minutes?) sive frames. its own. Such remarkable plasticity in

LOCOMOTIONIN A FRESHWATEROLIGOCHAETE WORM 441 tor for forward crawling is activated head counter-clockwise tail by touch in posterior segments, but only when worms are lying on a rough surfaceor enclosed by substrate.Some- direction of swimming how, these conditions provide the cues needed to "turn on" the generatorfor forward crawling, rather than the one for swimming. The opposing sensory fields for for- ward and rearward crawling, and for swimming and reversal, illustrate key transitions along segments with respect to neural connections between touch sensory neurons and centralpat- head tail direction of body wave tern generators. References Alexander, R.M. (1982). Locomotionof

clockwise Downloaded from http://online.ucpress.edu/abt/article-pdf/61/6/438/49035/4450725.pdf by guest on 25 September 2021 . New York: Chapman & Hall. Figure5. Diagramof hands-onmodels for helical swimming in Lumbriculus.Models Drewes, C.D. (1996a).Those wonderful may be made of solid-core, insulated wire. Both models show a helical wave in worms. Carolina Tips, 59, 17-20. about mid-body position. Drewes, C.D. (1996b). Heads or tails: Patterns of segmental regeneration in a freshwater oligochaete. In J.C. neural and behavioral functions has geous in open water conditions where Glase (Ed.), Tested Studies for Labora- obvious survival benefit. worms are unprotectedand have little tory Teaching, vol. 17, pp. 23-24. tractionto assist in escaping from both- Association for Biology Laboratory ersome stimulationto their tails. Swim- Education (ABLE). Discussion ming enables a worm to quickly move Drewes, C.D. (1999). Helical swim- a short distance, perhaps one or two ming and body reversal behaviors The most obvious movements dur- in (Family ing crawling are peristaltic waves of body lengths. Reversal behavior also seems advantageous in open spaces. ). Hydrobiologia, (in contractioncaused by circularand lon- press). gitudinal muscles. Though not seen Since a worm can't swim backwards, reversalenables it to rapidly reposition Drewes, C.D. & Brinkhurst, R.O. with the naked eye, crawling is also (1990). Giant nerve fibers and rapid assisted by movements of tiny bristles its head away from the stimulus threat. Then, if stimulated again, near its tail, escape reflexes in newly hatched (chaetae) along the body. Lumbriculus aquatic oligochaetes, Lumbriculus has four pairs of chaetae in each seg- it swims. In lab cultures, reversal and swim- variegatus (Family Lumbriculidae). ment. In shortened segments, chaetae InvertebrateReproduction and Develop- project outward, thus providing trac- ming sometimes occur when worms that are tightly packed around a rich ment, 17, 91-95. tion and preventing slippage due to Drewes, C.D. & Fourtner,C.R. (1990). pulling and pushing forces nearby. food source are suddenly disturbedby vibration or touch. After quick rever- Morphallaxis in an aquatic oligo- Movements of chaetae during under- chaete, Lumbriculus variegatus: Reor- water crawlingin Lumbriculusare simi- sal, each worm swims away from the ganization of escape reflexes in lar to those in earthworms (Gray & pack, creating a burst of body move- regeneratingbody fragments. Devel- Lissmann 1938). ments radiating in all directions from opmental Biology, 138, 94-103. Experiments I and II clearly show the food. It seems possible in nature Drewes, C.D. & Fourtner,C.R. (1993). how worms' responses to touch that this could startle or confuse a Helical swimming in a freshwater depend on environmentalcontext and predator and improve each worm's oligochaete. Biological Bulletin, 185, site of stimulation along the body. chance of survival. 1-9. Crawling responses to tail and head Though not known for sure, Lumbri- Gray, J. (1968). Animal Locomotion.Lon- stimulation are used when worms are culus probably uses different central don: Weidenfeld & Nicolsen. in confined spaces. Swimming move- pattem generators for crawling and Gray, J. & Lissmann, H.W. (1938). ments, on the other hand, are advanta- swimming. The centralpattern genera- Studies in animal locomotion VII. Locomotory reflexes in the earth- worm. Journalof ExperimentalBiology,

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POag...... Lesiuk, N.M. & C.D. om Drewes, (1999). -AS T ...... T...... Blackworms, blood vessel pulsa- ...... tions, and drug effects. TheAmerican Bruce Futh's''.of -2 Biology Teacher, 61(1), 48-53...... St Trueman, E.R. (1975). The Locomotion of Soft-bodied Animals. London: ''Ohn' Pitts of Th""".""e:P .t .-C Edward Arnold. Young, D. (1989). Nerve Cells and Ani- -October. . 3'O.. mal Behavior. New York: Cambridge ..... University Press.

442 THEAMERICAN BIOLOGY TEACHER, VOLUME 61, NO. 6, JUNE1999