Examine your petri dishes of regenerating planarians. If some of you proved to be excellent surgeons than you can consider using planaria as your project. The goal here is to cut the planarians various ways and then speculate on the distribution of neoblasts that would explain your results. What other factors (genetic) could be important? You will also have to do some library research to be able to fully explant your results.

Another alternate project for those interested involved Lubriculus from this lab and the testing of various drugs to obtain dose response curve and then compare results to those obtain with spiders for general classes of drugs (nerve stimulants and depressives). You will also have to do some library research to be able to fully explant your results.

General introduction and Clitellata

Don't you ever laugh as a hearse goes by,For you may be the next to die.They wrap you up in a big white sheet,And cover you up from your head down to your feet.They put you in a big black box,And cover you up with dirt and rocks.All goes well for about a week,And then your coffin begins to leak.The worms crawl in, the worms crawl out,The worms play pinochle on your snout.They eat your eyes, they eat your nose,They eat the jelly between your toes.A big green worm with rolling eyes,Crawls in your stomach and out your eyes.Your stomach turns a slimy green,And pus pours out like whipping cream.You spread it out on a slice of bread,And that's what you eat when you are dead.

In this lab, because specimens are expensive, you will be sharing larger specimens. To watch animals feed you will need to place food in containers. There is a danger of fouling the containers. Please be careful and add only the amounts specified in the instructions to your containers. If a container looks foul to you, call the lab instructors attention to it so she can add new water.

If she instructs you to do so, make sure you know whether you are to add spring or salt water to your bowl. A rule of thumb is that Clitellata (earthworms and relatives) are terrestrial or fresh water forms, Polychaetes are salt water forms and so are sipunculids and echiurans. We have plenty of the microscopic fresh water forms, so these can be dumped when you are done. It will ensure that the rest of the population does not suffer.

Background: Annelida consists of the segmented worms in the major classes Polychaeta (bristleworms), and Oligochaeta (earthworms and relatives), with a total of about 12,000 known species in marine, freshwater, and terrestrial environments.

Most annelids have chitinous bristles, or chaetae, secreted by epidermal cells, that project from the body. The body wall consists of a collagenous cuticle secreted by the monolayered epidermis. The coelom is large, segmentally compartmented, lined by peritoneum, and well developed in polychaetes and oligochaetes but reduced in leeches. Successive coelomic spaces are separated by septa which consist of double layers of peritoneum with connective tissue in between. The right and left sides of each segmental coelom are separated by longitudinal mesenteries which, like septa, are double layers of peritoneum with connective tissue between.

The gut is a straight, regionally specialized tube that begins at the mouth at the anterior end and extends for the length of the body to end at the anus on the pygidium. It penetrates each septum and is supported by dorsal and ventral mesenteries. Like that of most invertebrates, the gut consists of ectodermal foregut, endodermal midgut, and ectodermal hindgut. The nervous system consists of a dorsal brain in or near the prostomium, a pair of circumpharyngeal connectives around the anterior gut, and a double, ventral nerve cord with paired segmental ganglia and nerves. The circulatory system of most annelids is a set of tubular vessels, some of which are contractile and serve as hearts. The circulatory system is absent or greatly reduced in leeches. The system includes a dorsal longitudinal vessel above the gut in which blood moves anteriorly, a ventral longitudinal vessel below the gut, in which blood moves posteriorly, and paired segmental vessels that connect the dorsal

and ventral vessels. The digestive, circulatory, and nervous systems are continuous and pass through the segments.

Respiration is accomplished in a variety of ways. In some, the general body surface is sufficient but gills are present in most polychaetes, many leeches, and a few oligochaetes. Excretory organs are metanephridia or protonephridia and typically one pair is present in each segment. These osmoregulatory organs are best developed in freshwater and terrestrial species. The sexes are separate in polychaetes but oligochaetes and leeches are hermaphroditic. In the ancestral condition paired clusters of germ cells were present in each segment and released developing gametes into the coelom. In derived taxa reproductive functions tend to be confined to a few specialized genital segments. Gametes mature in the coelom or its derivatives and fertilization is external. Gametes are shed through ducts derived from metanephridia or by rupture of the body wall. Spiral cleavage follows fertilization. Clonal reproduction is common.

Clitellata

Clitellata, the sister taxon of Polychaeta, includes earthworms and their allies. The head is reduced and its sensory functions minimized. Clitellates lack the parapodia characteristic of polychaetes although chaetae may be present. Also lacking are head and pygidial appendages. Present however, is a girdle-like band of secretory epidermis, the clitellum, near the anterior end of the worm. The brain has moved out of the prostomium into a more posterior segment. These worms are hermaphroditic and have gonads and reproductive equipment restricted to a few specialized genital segments. Development is direct, without a larva.

In the laboratory, you will find several living representatives of clitellate annelids. You will use the earthworm as the model organism. Earthworms are also know as oligochaetes, a clade of clitellate annelids that are very common in the United states. Please compare the

other living representatives to the living earthworm with regard to overall anatomy, feeding and locomotion.

1. External Anatomy and locomotion: Obtain an earthworm, Lumbricus terrestris. Place it in a large dish to study its external anatomy and locomotion.

Orientation

Examine the external features of the worm . The anterior end is usually larger than the posterior and is round in cross section whereas the posterior tends to be flattened dorsoventrally

A dorsal view of the anterior end of Lumbricus. The segments are numbered.

Clitellum

A band of thickened secretory epithelium, the clitellum (clitell = saddle), girdles the body near the anterior end. The clitellum secretes a mucous cocoon, into which gametes and albumen are released and where fertilization occurs,

Segments

The body is segmented and each segment is separated from its neighbors by a distinct circumferential groove. The anteriormost true segment is the peristomium (peri = around, stome = mouth). Anterior to the peristomium is a small lobe, the prostomium (pro = before) that, for embryological reasons , is not considered to be a segment. The peristomium encircles the large, ventral mouth and is an almost complete ring around the body. The prostomium fits into a small, dorsal notch in the peristomium. The prostomium is a small lobe dorsal to the mouth and anterior to the peristomium. The segments are numbered anterior to posterior beginning with the peristomium.

Count the preclitellar segments in your specimen. ____________ How many segments contribute to the clitellum? _____________ Compare your counts with those made by other students. Does the number of segments in each region appear to be constant or variable? ____________

The segments posterior to the peristomium are complete rings, without notches, and extend uniformly for the entire length of the worm. The posterior most division of the body is the pygidium, which encircles the anus at the posterior tip of the worm. Like the prostomium, the pygidium is not considered to be a true segment.

Chaetae or SetaeThe eight small chaetae or setae on each segment (except the first) are usually visible with adequate magnification (25X). Chaetae or setae are small chitinous bristles emerging from pores in the integument on the ventral half of the worm. The chaetae are arranged in four pairs, two on each side . Each segment has a pair of lateral chaetae and a pair of ventral chaetae on each side. The chaetae are used as anchors when burrowing to hold parts of the worm against the so that elongation of the animal results in controlled, usually forward, motion. A ventral view of Lumbricus. The worm has been rolled slightly to reveal part of the right side.

The chaetae are equipped with protractor and retractor muscles and are retractile. In your specimen they may be withdrawn in some, or even all, of the segments. Careful examination,

however, should reveal some segments with extended chaetae. Chaetae are amber or brown, slightly curved, and short.

Rinse your worm gently with tap water and hold it in your hand on the stage of the dissecting microscope. Focus on the lateral body wall and watch the chaetae. You may see the animal retract or protract some of its chaetae while you watch. Place it on a clean dish, and watch it move. Does it seem to have difficulty moving on the smooth glass surface. Place some stones and dirt in the dish and now watch it move. a. Explain why it was difficult for the earthworm with a muscle that essentially act to distort a hydroskeleton to move on the glass dish but not the dish that contains dirt and stones. You may use the notes below, which are a summary of lecture notes, to help you draft your explanation. Record your observations in your notebook. b. When the earthworm moves in dirt do any segments move as a unit? c. Does the wave of contraction and relaxation move from front to back or back to front?

Annelida (earthworms)Annelid worms are excellent examples of a hydrostatic skeleton in the form of a coelom in action.

In earthworms, the hydrostatic skeleton or coelom is surrounded by sheaths of longitudinal and circular muscles. When the longitudinals shorten a short fat animal is produced, when the circulars contract a long thin one.

Locomotion begins with a contraction of the circular muscles in a limited region of the anterior end of the body. This contraction passes backwards down the body as a peristaltic wave, each wave being followed by a wave of contraction of the longitudinal muscle; . This continues in regular alternation.

At those regions where the longitudinal muscle is contracting the body surface bulges outwards and the chaetae are protruded. At these points, the worm can exert a thrust against the substratum. Where the circular muscle is contracting the body becomes thinner, and the chaetae are withdrawn. Here the segments extend forwards, aided by the thrust from the swollen regions. Thus the extension of the thinner parts of the body can be translated into forward movement.

Peristaltic movements of this sort can be used to drive the animal along and if, as if earth worms, some form of anti-slip device is incorporated into the design, considerable forward pressure can be generated. The importance of the thrust against something like a burrow wall and anti-slip device can be shown by placing the worm on a glass plate or other slick surface. Although the hydroskeleton again gives effective movement in a viscous media (a burrow), it proves ineffective for true locomotion on land.______________________________________________________________

Comparative external anatomy and movement in the Annelids. There are two alternate ways to proceed. 1. You may be asked to look at larger representatives of Clitellate and Polychaete annelids and then examine the effects of size in some Clitellates. 2. Or you will be asked to finish your clitellate work and then proceed to examine Polychaetes and their relatives.

2. Hirudinea : Leeches belong to another clitellate annelids. We only have 3 specimens, so when you are done pass your specimens on. Leeches are also clitellad annelids, but modified for an ectoparasitic life style. The body has a terminal posterior sucker and a dorsal subterminal anus. An anterior sucker is present in all but one species (Acanthobdella peledina).

As always make sure you record your observations in your journal.

Obtain a living leech and place it in some fresh water. Allow the leech to remain undisturbed for about 5 minutes in a well lighted room. Pass your hand over the container so that a shadow falls on the leech. a. Observe and record the leeches' actions.

b. Allow the leech to remain undisturbed for at least 5 minutes. Use a plastic ruler or tongue depressor to stir up the water surface. Observe and record the leech’s actions. What parts of the leech are receptive to water movement? Why would leeches have organs that are sensitive to water vibrations?

c. Watch it move and compare its movement with that of an earthworm. Do you feel the leech relies on its hydrostatic skeleton as much as an earthworm? Do you see types of movement, you would not expect in animal such as an earthworm without the “compact” body of a leech.

d. Finally examine the leech’s external anatomy. Does it have more or less segments than an earthwork. Can it be divided into the same three areas? Does it have chaetae?

e. Add a snail or small chunk of chicken livers to your dish. Place it on the side of the dish furthest from the leech. How does the leech react? Watch your leech fed. If they are hungry enough they will feed on the beef even if they prefer snail. If your specimen is cooperative, you

will develop a respect for just how flexible a body a leech possess.

__________________________________________________________________Determine with your instructor’s counsel to continue looking at other members of this group or move to the polychaetes. Everything depends on what polychaetes are sent and whether we think we can keep them for another week. We can always order more freshwater and terrestrial Clitellates. _____________________________________________________________3. Lumbriculus variegates is a detritus feeder like the earthworm and a perfect example of severe miniaturization of form.

As always make sure you record your observations in your journal.

a. Obtain a few mudworms. Place them is a small Petri or observation dish. Note how this oligochaete moves. Also take one or two and put them on a wet paper towel. Note the difference in “swimming” locomotion versus locomotion in an environment were they can contact surface.

b. You may be able at the highest power of your stereoscope to focus and watch them feed. Use the fish food provided. Add a few tiny tiny crushed pieces of one flake, or one flake to the bowl. As always record your observation. Notice how transparent at high power these worms are.

These worms are often used for experiments that look at the effects of drugs on cardiovascular function.

Place your worm in a plastic pipette and let it move to the tip. Then place the tip of a capillary tube close to the tip of the pipette and gently squeeze. The worm should move into the capillary tube, which you can place then in a small observation dish. Observe the internal anatomy

of the worm. Can you trace blood flow from dorsal to ventral? Time dorsal vessel contraction rate.

c. Take a video or photographs of blood flow showing the changes in vessel diameter. Any evidence for how these animals respire?

Then add a bit of alcohol (70 % ETHANOL ONLY-Alcohol is very toxic to invertebrates so start with 1ml or less in a dish 6 inches in diameter. ) to bowl or simply place your bowl on an ice block. d. What is the effect on heart rate? Describe the effect, noting whether you used ice or alcohol in your notebook. If we had more time in this lab, we could test a host of other drugs and even determine a dose response curve. Do not place any worms exposed to alcohol or cold back in the colony dish. Place them in a separate dish until we can determine if they will recover and not die and foul the colony dish..

4. Various small annelids. Examine several small species of annelids to document the effects of size on segmentation and the types of organs found in these species.

Below are some photographs of some of the forms available.

Dero, back end, high power Yes that is the hind end, not the head, so what is that claw like structure?

Aeolosoma-often multiplies by fission, can get very long worms with several generations developing behind the parent generation. So, are your cultures reproducing asexually?

Stylaria, Note large setae and prominent feeding structure at anterior. So if that is what they use to feed, how do they feed?

Enchytraeus. Is a small terrestrial worm. . Does it show the same effects of miniaturization that you sre seeing with one the small freshwater forms? Enchytraeus is often used for toxicity testing.

Pick two forms, identify from the pictures which two forms you picked. a1 and a2 Photograph your two forms including their names in a corner of the photographs. b1 and b2. Compare them to Lubriculus in overall morphology and anatomy (number of segments, visibility of segments, presence or absence of circulatory vessels, etc.). Make sure you take some of the green stuff in the culture with you, so you can watch them feed as well.

Below are some urls that may help you in identifying anatomy particular to a species, but do not simply download the photos or videos as your own. We will know!!!www.microscopyu.com then go to galleries then pond life

http://www.micrographia.com/specbiol/helmint/annelhom/olig0100.htm#oligolinkThe first few unidentified pics are probably aeolosoma.

http://www.microbelibrary.org/index.php/library/resources/3190-aeolosoma-hemprichi-ingestion-of-soil-particles-and-digestion-of-microbial-films

What general trends do you see? Record your observations in your journal.

c. Produce a movie of locomotion in one of these species. How does the locomotion of your choice vary from that shown by Lubriculus and the earthworm.

To examine these more closely, you may need to watch them under a depression slide or under slight compression on a regular slide. Only use low power, 10x or 20x so the slide can still hold enough water for these to move.