HEADS OR TAILS?
Investigating Patterns of Regeneration in Freshwater Oligochaetes


Punch cartoon by Linley Sambourne 1882.
 

I. Introduction
Embryonic development is an orderly and stereotyped process during which an organism's cells differentiate and its body gradually acquires adult-like characteristics. If the adult organism is bilaterally symmetrical (as in many invertebrates and all vertebrates), then at some time during embryogenesis a body plan is established along three different body axes: (1) an anterior-posterior axis that establishes head and tail ends, (2) a dorsal-ventral axis that establishes front and back sides, and (3) a left-right axis. During development, the fate of differentiating cells and tissues will vary, depending on their exact position within these axes. Cells closest to the head end will be influenced by chemical factors (called morphogens) and by physical constraints to develop proper, head-like features. Cells in the middle will be similarly influenced to develop features appropriate to a mid-body position, etc. This developmental process of acquiring a characteristic body plan with position-specific features is referred to as pattern formation.

In some invertebrates (such as hydra, planaria, and certain segmented worms) the process of pattern formation may also be played out during the regeneration of body parts. Loss of body parts, such as a head or tail, can occur following accidental damage or predation, or it can occur normally in conjunction with certain types of asexual reproduction, one of these being fragmentation. Fragmentation is a self-produced, mechanical breaking of the organism into two or more pieces, followed by regenerative growth of missing parts in each fragment.

If a worm loses a part of its posterior end by fragmentation, how does the worm "know" whether to regrow a new head or tail at the wound site? How does it "know" how long the replacement part should be? In relatively large and segmented organisms, such as annelid worms, these questions don't have clear answers because underlying mechanisms of development are not completely understood. However, we can begin to understand these mechanisms if we carefully observe the regeneration process following various surgical manipulations and then try to determine the "rules" and general patterns that govern the process.

When worms regrow missing body parts, one of two general regeneration patterns seem to be followed. One pattern is segment regeneration by compensatory growth. That is, the number of regenerated segments exactly equals the number removed. So, for example, if five head segments are amputated, then exactly five segments head segments regenerate in their place. Thus, with this pattern, all newly regenerated segments acquire precisely the same positional and numerical identity as the segments which were removed.

An alternative pattern is that the number of regenerated head segments is a constant. So, regardless of whether 5 or 25 head segments are amputated, only a fixed number of new head segments regenerates in their place. If this happens, then a developmental reorganization of the segments adjacent to the new head also occurs. That is, adjacent segments become transformed anatomically and physiologically to match their new positional identity along the body axes. This reorganization, which can occur without cell division, is called morphallaxis.

In this exercise you will determine which general pattern of segmental regeneration (compensation or morphallaxis) occurs in the freshwater oligochaete, Lumbriculus variegatus. One key experiment in making this determination is to systematically compare head and tail regeneration in amputated fragments that have approximately the same number of segments but differ with respect to their original position within the longitudinal body axis. That is, you will compare patterns of head and tail regeneration in anteriorly derived fragments with patterns in posteriorly derived fragments of approximately the same length. You may also determine whether short and long fragments from the same body region have differing capacities for head and tail regeneration, and whether there is a minimal size fragment that can support head and tail regeneration. (Refer to Appendix A and Appendix B for general background information and biological facts about Lumbriculus).

IMPORTANT PRECAUTIONS:
(1) During culture and handling, never expose worms or worm fragments to chlorinated tap water. They will die quickly. Use only de-chlorinated water. To de-chlorinate, let water sit in a shallow. open container for at least 48-96 hours. Continuous bubbling will speed up the removal of dissolved chlorine.
(2) When handling worms, avoid using pipettes with sharp or ragged edges that could damage worms. Always use an eyedropper or plastic disposable pipette to pick up and transfer worms or worm fragments. Never squeeze, pinch, or press on worms. They are very fragile. Never handle the worms with forceps.

II. Materials needed for regeneration experiments
● medicine dropper or disposable plastic pipet
● aged, dechlorinated tap water
● new, single-edge razor blade, or "fake blade" (= 1" long strip cut from edge of acetate sheet; use straight edge of strip as blade)
● filter paper discs or squares
● disposable petri dish (or other shallow, flat container) for cutting fragments
● dissecting microscope with illuminator (side lighting, if possible)
● thin plastic ruler with millimeter markings
● black paper (provides dark background under dishes)
● small labels or label tape
● fine marking pen
● live Lumbriculus variegatus - ideally 3-5 worms per student. Worms are sold in bulk in pet shops and tropical fish stores as "California blackworms" that are used as live fish food. [SEE Appendix C for instructions on establishing lab culture of worms.]
● numerous small containers for maintaining individual fragments as they regenerate
[NOTE: Possible containers include very small petri dishes with covers, 6-well or 24-well tissue culture dishes with covers, or 1-2 ml capped disposable centrifuge tubes. One student may create numerous fragments each requiring a separately labeled container with tight-fitting cover.]

III. Procedures for handling and cutting worms
Place a piece of filter paper in a plastic petri dish and saturate the paper with dechlorinated tap water (Fig 1A). Using a dropper or pipet, transfer a worm to the middle of the paper.  If the worm sticks to the inside of the dropper, draw more water into the dropper and gently shake the worm towards the tip so it can be readily expelled. Next, tilt the petri dish to one side and withdraw excess water, leaving the worm about in the middle of the paper. Observe the worm's movements in the petri dish. Identify the head end of the worm. To do this, you may need to use a dissecting microscope and the following clues:

● the head end is blunter and more darkly pigmented in comparison to the more slender and paler tail end .
● the head end is usually more active in terms of searching movements and locomotion than the tail end.

Remember that the object is to isolate worm fragments of known length and origin, and then study regeneration in each fragment. One strategy is to cut the worm into six or eight pieces of equal length. Segment regeneration is then observed at the cut end(s) of each fragment. Wait until the worm is approximately straightened and then position the razor blade above the worm about 1/8 of the way back from its head end (Figure 1 B). [Note: the blade edge should be held parallel to the surface of the dish but crosswise to the worm's body.] Quickly press the blade through the worm and against the paper, holding the blade down for a couple seconds. The worm should separate into two pieces with little or no bleeding.

Figure 1. Procedure for obtaining and isolating individual fragments (below).
(A) A mudworm is ejected from a pipet into a petri dish containing filter paper saturated with aged tap water.
(B) When straightened, a fraction of the worm's body is cut off with a razor blade.
(C) Aged tap water is ejected from the pipet, thus flushing the fragment into a pool of water at the edge of the dish.
(D) The fragment (arrow) is drawn up into the pipette and transferred to a storage container.

Now, remove the short, head end from the dish by flushing it off the paper and toward the side of the dish using a few squirts of dechlorinated tap water from the eyedropper (Figure 1 C). Draw up this piece into the eyedropper (Figure 1 D) and transfer it to a labeled tube or container (Label it 1/8 to indicate that this is the first eighth). Now, find the anterior end of the remaining worm and cut off another piece about equal in length to the first piece. Put this fragment in a separate container labeled 2/8 and repeat for 3/8, 4/8 fragments, etc.

After each eighth has been obtained and transferred to its container, add dechlorinated tap water until the tube is about half full. Store all containers together in the dark (at 68-74 degrees F), keeping them tightly and continuously covered to avoid evaporation and spillage. Do not add any food to containers while regeneration is in progress.

Begin repeated observations of regeneration in each fragment, starting at weekly or twice-weekly intervals for the next 3-4 weeks (Refer to Appendices D, E, and F for examples of results and sample data sheets). Always make sure dishes remain covered to prevent evaporation; add dechlorinated tap water, if needed. Observations of the fragments should be done by removing worms from their tubes and inspecting under the dissection microscope with a minimum of handling and direct touching. Placing them on saturated black paper may help improve visual contrast and may make counting of segments easier.

IV. Suggested projects and questions:
(1) Compare head and tail regeneration in each piece. What new body part(s) did the 1/8,2/8...8/8 pieces regenerate? Can you tell a new head from a new tail? How? See if you can count new head and tail segments. Or, use a millimeter ruler to measure the length of the growing head and tail. A flat, transparent ruler placed under the dish works well to make make these measurement estimates.

(2) Describe and draw new and old segments during head and tail regeneration. Include features such as segment shape, pigmentation, and blood supply. [Note: Even before regeneration begins, the head end of any worm fragment can be told from the tail end by noting that blood always pulsates in a tail-to-head direction in the worm's dorsal blood vessel ].

(3) Did all fragments survive? Which ones did not? Give possible reasons why some pieces died. Pool results from the entire class to see overall trends in fragment survival.

(4) Do fragments from more posterior origins have a greater or lesser capacity for tail regeneration than fragments from more anterior origins?

(5) What about the capacity for head regeneration in these fragments? KEY QUESTION: Do your results support the idea that Lumbriculus regenerates by compensatory regeneration or morphallactic reorganization?

(6) What is the smallest fragment that can survive and regenerate? To study this question, you may want to use another worm, subdividing it into even smaller pieces. Do small fragments regenerate longer or shorter tails than larger fragments?

(7) Did any fragments show abnormal regeneration and produce any monstrosities?

Appendix A: Background and references
Lumbriculus variegatus, the species used in this exercise, should not be confused with Tubifex, or other tubificid worms, whose regenerative powers are not nearly so great as Lumbriculus. Despite its potential for regeneration studies, Lumbriculus is not widely available from all biological supply companies. Instructors can gain access to these worms in a number of ways, as described here: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/WORMSO5.htm
Worms are available for retail purchase in large tropical fish or pet stores, where they are called "California blackworms." They are usually sold in bulk for living fish food. Second, it may be possible to locate natural populations of Lumbriculus near your residence or school, since it is widely distributed throughout North America and is especially abundant in the leaf litter along the shallow margins of marshes and ponds. Third, live specimens may be obtained from others who have established lab cultures of them. It is easily cultured and large populations may be established within a couple months from small numbers of stock worms. If you have additional questions relating to sources, field collection, laboratory culture, or biology of Lumbriculus, please feel free to contact C. Drewes. 

Web references:
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/
http://www.carolina.com/tips/worm/worm.asp
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/WORMSO5.htm
http://www.eeob.iastate.edu/facultv/DrewesC/htdocs/LVCULT.htm
http://www.eeob.iastate.edu/facuIty/DrewesC/htdocs/Lvgen4.PDF
http://www.eeob.iastate.edu/facuIty/DrewesC/htdocs/lvreqencolor.JPG
http://www.zoo.utoronto.ca/able/volumes/vol-17/2-drewes.pdf

Barnes, R.S.K. et al (2001) The Invertebrates: A New Synthesis, Blackwell Science Ltd. [ISBN: 0-632-04761-5]

Drewes, C.D. and C.R. Fourtner (1990) Morphallaxis in an aquatic oligochaete, Lumbriculus variegatus: Organization of escape reflexes in regenerating body fragments. Devel. Biol. 138:94-103.   

Drewes, C.D. (1996)  “Heads or Tails: Patterns of Segmental Regeneration in a Freshwater Oligochaete” Association for Biology Laboratory Education (ABLE), Volume 17 in Tested Studies for Laboratory Teaching, Edited by J.C. Glase, pp. 23-34.

Appendix B: Biology Facts about Lumbriculus
    Classification: Phylum name- Annelida   Class name- Oligochaeta
    Genus and species- Lumbriculus variegatus
    Common name- mudworm, or California blackworm

Habitat and Ecology:  These worms live in muddy sediments, especially in shallow water along the edges of marshes and ponds throughout the United States, and many other parts of the world. See:
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/gullpt.jpg
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/worm-habitat.JPG

Body and support:  Mudworms usually have 150-250 body segments. They have no rigid skeleton, but the fluid inside their body gives them support and form (hydrostatic skeleton).  See: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/Lv-KSNr.jpg

Reproduction and Development:  Like earthworms, each mudworm has both male and female sex organs, but sexual reproduction appears to be uncommon. Reproduction is more common by asexual fragmentation. Worms simply break apart and each fragment becomes a new worm by growing a new head and/or tail. This process of re-growth is called segmental regeneration. See: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/lvregencolor.JPG

Feeding/Digestion:  Worms eat small living and dead material in mud. They have a complete digestive tract with a mouth and anus. Contractions of the intestine are easily visible in whole worms.

Respiration:  Worms have no obvious respiratory organs. They respire through their skin, and mainly across the top surface of their tail. If the water is not too deep, worms will often stick their tail up to the water surface to obtain more oxygen. Worms can survive for long periods with very low amounts of dissolved oxygen in the water. See: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/lvtails.jpg

Circulation:  Blood flows in blood vessels and capillaries. The dorsal blood vessel is easily seen in each worm. This vessel pulsates rhythmically, pumping blood throughout the body. Blood always flows in a forward direction (from tail-to-head) in the dorsal blood vessel. The large ventral blood vessel does not pulsate. Blood is red due to hemoglobin. See:
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/DBV-Lv.gif
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/DBV.JPG
http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/looking2.JPG

Nerves. muscles. and movement:  A nerve cord, made up of many nerve cells, is found just below the intestine, along the entire length of the body. Segmental nerves arising from the nerve cord innervate the worm's skin and body wall muscles. Worms use their muscles to crawl through the mud. They get extra traction from bristles (chaetae) on the side of their body. See: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/anima-peristalsis.gif
Worms rapidly shorten their head or tail end when it is touched; these reflex responses are triggered by impulses in giant nerve fibers within the nerve cord. Worms holding their tail up to the water surface use photoreceptors in their tails to sense moving shadows. When a shadow is detected worms rapidly withdraw their tails from the surface; this response is also triggered by impulses in the giant nerve fibers. In water containing little substrate, worms can also swim for short distances by making corkscrew movements with their body. See: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/anima-Lv.gif

Appendix C: Starting a colony of worms in the lab:
MATERIALS NEEDED:
• aquarium (maximum = 5-10 gallon; minimum = gallon-sized fishbowl; or large Tupperware container)
• brown paper towel
• sinking fish food pellets
• distilled water (no chemical additives)
• aquarium pump with tubing and adjustable air flow
• live Lumbriculus variegatus (sold in bulk in pet shops and tropical fish stores as "California blackworms")
• medicine dropper or plastic pipet
• aged, dechlorinated tap water (or spring water)
• turkey baster

To start an asexually reproducing colony of worms, add the following to an aquarium: (1) aged, dechlorinated tap water (half full); (2) thin strips of brown paper towel, to lightly cover bottom; (3) a few hundred worms. The aquarium should be continuously aerated by gentle bubbling. No filter is necessary. Add an amount of food which can be completely eaten within a day; look for its disappearance. For a few hundred worms this may be approximately one or two fish food pellets every few days. Do not overfeed. Water lost to evaporation should replaced by adding distilled water, rather than aged tap water. This prevents build-up of mineral content in the aquarium. After a few weeks, when the paper towel disintegrates, new strips may be added periodically. If the aquarium water becomes cloudy or yellow, it may be partially drained by siphoning and replaced by adding clean, aged, dechlorinated tap water. Worms should now reproduce continuously and survive indefinitely. As the colony becomes larger, its oxygen demand increases, so it is critical that aeration is continuous or there may be mass die-off. Also, it is possible that the colony can eventually become too large. Surplus worms can be removed and used as live food for other animals (e.g. fish) or as "starters" for worm colonies in additional aquaria. To remove a large number of worms at one time from the aquarium, allow worms to collect near a food pellet and quickly withdraw them using a large bore pipette, or a turkey baster. These can be transferred to a container of aged tap water.

Appendix D.  Examples of head and tail regenerates

(A) Head and tail regeneration after three weeks in a 28-segment-long fragment, originating from the posterior half of a donor worm. Original segments are indicated between the two dots. Eight new head segments were regenerated on the anterior end of the fragment; this is the usual number of regenerated head segments in either anterior or posterior fragments of all lengths.
(B) Head and tail regeneration after three weeks in a six-segment-Iong fragment, originating from the anterior half of a donor worm. Original segments are indicated between the two dots, a distance of about 2.5 mm.
(D) An abnormal anterior regenerate with two heads. The regenerated anterior end consisted of five common segments and three segments in each of the two head branches. Demarcation between old and new segments is shown at the white dot.

The diagram below shows a typical progression of head and tail regeneration in a f5-segment-long Lumbriculus fragment, beginning with blastema (bud) formation at each end of the fragment. Note that delineation of new segments occurs relatively rapidly and quickly. Note also that growth of new head and tail segments occurs at the expense of the original fragment, which is undergoing 'de-growth.' However, the 5 original segments remain clearly delineated and distinct from all newly regenerated segments. That is, each original segment is longer and more darkly pigmented than newly regenerated segments.

Appendix E. Tail segment regeneration in Lurnbriculus.
The graph below shows the relationship between the number of regenerated tail segments and the length of fragments. All fragments originated from posterior regions of donor worms.



Appendix F. Mudworm regeneration timetable and data sheet
Timetable:
Day 0:  Cut worm into fragments. Label and store fragments.
Day 7:  Check to see if fragments are alive. Note blood flow pattern in fragments to determine head and tail ends. Assess degree of head and/or tail regeneration, as per data sheet below:
Day 14:  Record results, as on day 7.
Day 21:  Record results, as on day 14.
Day 28:  Record results, as on day 21.

Data sheet
WORM NUMBER / NAME_____________________________________  DAY______________

Sketches of regenerated head and/or tail end.
Determine length (in mm) and/or number of new segments in regenerated head/tail ends
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Fragment 1/8

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Fragment 2/8

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Fragment 3/8

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Fragment 4/8

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Fragment 5/8

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Fragment 6/8

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Fragment 7/8

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Fragment 8/8

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