ANNELID LAB Phylum Annelida Class Oligochaeta 1. Lumbricus terrestris preserved specimen for dissection 2. Lumbricus terrestris cross section slides 3. Lumbricus terrestris longitudinal, whole animal slides Class Polychaeta 1. Nereis virens preserved specimens for dissection 2. Nereis virens prepared cross sectional slides 3. Nereis virens parapodial slides 4. Preserved demonstration specimens with morphological differences to examine/determine lifestyles Class Hirudinea 1. Hirudo sp. preserved specimens for dissection (be very careful, this is a difficult dissection) 2. Preserved demonstration specimens 3. Live leeches to examine movement
Phylum Annelida The annelids or segmented worms are characterized by an elongated body, divided into segments and formed on the plan of a tubular jacket of muscle surrounding a fluid-filled coelom. Although lacking a rigid internal skeleton. annelids can use the hydrostatic pressure of coelomic fruit, acted upon by the muscular body wall, as a "fluid skeleton," aiding in extension and flexing of the body in crawling, swimming, and burrowing. Locomotion in some annelids is also aided by numerous fine chitinous hairs, called setae, which project from the sides of the body. The annelid body plan has proven to be extremely plastic and adaptable in an evolutionary sense, so that we find a great diversity of form, habitat, and life style within the phylum. Three annelid classes are generally recognized: 1) OLIGOCHAETA have no parapodia, contain a clitellum (generally year round) and few setae (as the name implies). They include the familiar earthworms, as well as many marine and freshwater representatives. 2) POLYCHAETA have many setae (hence the name) and lateral body wall lobes called parapodia, as well as a diversity of tentacles and gill-like devices. Polychaetes are almost entirely marine, exceedingly diverse in form, and very numerous, comprising about 60 families and some 1600 genera. Archiannelida, a small and poorly known marine group, may be regarded as an order or subclass of Polychaeta rather than as a distinct class. Archiannelida are small worms that show a curious assortment of features, including weak segmentation, poorly developed parapodia, and frequent lack of setae. Some of these features may be primitive, others reduced. 3) HIRUDINEA or leeches have no setae or parapodia, possess a clitellum (however, not year round) and are found mainly in fresh water. Annelids are typically bilaterally symmetrical, elongate in an anterio-posteriordirection, cylindrical in cross section, and divided externally by a series of rings into body segments (metameres). The external segmentation is reflected by the arrangement of the internal organs, which are serially repeated. This condition is known as metamerism and is particularly pronounced in primitive polychaetes. Metameric segmentation distinguishes the annelids from other worm-like forms. It provides a degree of plasticity in that certain segments can become modified and specialized to carry out specific functions, and segments can respond either individually or collectively. The annelid gut is a straight tube supplied with its own musculature, so that it functions independently of muscular activity in the body wall. Like nematodes, annelids have a fluid-filled internal cavity separating the gut from the body wall which is similarly involved in locomotion as a hydrostatic skeleton. However, this space is not embryonically derived from the blastocoel as with nematodes, but is formed much later in development as a split in the mesoderm by which it is entirely lined (schizocoely). This fluid-filled internal space is a true coelom as found in arthropods and chordates. The excretory and circulatory systems are well developed, and some members of the phylum have respiratory organs (gills). The nervous system is concentrated anteriorly (cephalization) into cerebral ganglia from which arises a ventral nerve cord with segmental ganglia. The nervous system coordinates activity among segments In today's laboratory, we will examine the functional morphology of representative annelids from each of the three classes:
CLASS OLIGOCHAETA Examine the external features of the common earthworm Lumbricus terrestris, which lives in moist soil, remaining in its burrow during the day but emerges from its burrow during the evening or after it rains. Note the well-marked external segmentation. Locate the nonannulated segment (the clitellum) functioning as an attachment point during copulation and in the formation of egg cases or cocoons. Observe the subterminal mouth. The preoral area is called the prostomium (pro, before; stoma, mouth; GK). The first true segment of the body surrounds the mout and is called the peristomium (peri, around; stoma, mouth; GK). Locate the anus at the posterior end of the body. Note the presence of bristles (setae) on the body of Lumbricus. Describe the distribution and orientation of the setae. Are they evenly distributed on the dorsal and ventral surfaces? Procedure: Get a Lumbricus specimen for dissection. The entire surface of Lumbricus is covered by a cuticle. Strip the cuticle from segments 9-15 and, using the dissecting scope attempt to locate the external genital openings. Oligochaetes are hermaphrodites (monoecious). The paired vase deferentia open latero-ventrally on the fifteenth segment, and seminal grooves lead back from them to the anterior edge of the clitellum. During copulation, sperm pass along the grooves to the spermathecae of the partner worm. The spermathecal openings are located between segments 9 and 10 and 10 and 11 at the same level as the dorsal setae. The oviducts open on segment 14. Attempt to locate the nephridial openings on one or two of the stripped segments. These openings are dorsal to and in front of the ventral row of setae in all segments except the first three and the last. What is their function? Middorsal coelomic pores open from the coelom in the segmental grooves of all segments following 10, but are difficult to see. In life they exude coelomic fluid and help to keep the worm moist. Now examine the internal structure of Lumbricus. Anchor your specimen to the pan by means of a pin through the prostomium, and make a dorsal longitudinal incision with a pair of scissors (starting at the clitellum). Be careful not to injure the internal organs when making the incision. Using forceps and a dissecting needle, open the cut and free the body wall from the septa. Note how the septa are segmentally arranged and how the gut is held in place by septa. Once your worm is opened, pin the body wall to the pan to fully expose the internal structures. The muscular pharynx is the principal organ of digestion; it narrows posteriorly to form the esophagus, which is surrounded by the five
pseudo-hearts or aortic arches of the closed circulatory system. Can you detect the cerebral ganglia and longitudinal nerve cords? In segment 10 is a pair of small diverticula, the esophageal glands which secrete digestive enzymes. In segments 11 and 12, similar pairs of esophageal glands contain calcium carbonate particles and have a whitish color. These calciferous glands are excretory in function and appear to function in controlling the level of calcium ions and ph in the blood and coelomic fluid. Posterior to the esophagus is a thin-walled crop (segments 14-16) which temporarily stores food as it is being digested and the thick-walled gizzard (segments 17-19) which physically masticates the food. The remainder of the gut is the stomach-intestine where the products of extracellular digestion are absorbed. This area is covered by yellowish
chloragogue cells where glycogen synthesis, protein deamination, and the formation of ammonia and urea occur. Oligochaetes such as Lumbricus have a fairly well-developed blood vascular system with main pulsating contractile vessels, and capillary networks supplying the gut, body wall, and other organs. Locate the dorsal and ventral blood vessels of Lumbricus and the pseudo-hearts. You can see blood pulsating in live earthworms when using the dissecting microscope. Describe the blood movement and location of the blood vessels. The red color of the blood is due to the presence of hemoglobin, which is not carried in cells, but is free in the blood and coelomic fluid. The excretory organ found in most annelids is the nephridium. In Lumbricus, almost every segment contains a pair of nephridia. Examine different cross-section slides in order to find a well preserved nephridium. Each consists of a ciliated funnel, the nephrostome, which communicates with the coelomic cavity and is situated posteriorly in the segment. A duct leads from the nephrostome, passes through the intersegmental septum posterior to the nephrostome, and continues as a fine ciliated coiled tubule surrounded by blood capillaries. The tubule leads to a bladder that discharges to the exterior via a nephridiopore, which opens near the ventral pair of setae. The cilia of the funnel and tubule beat and draw fluid from the coelom; wastes and other materials are exchanged with the blood in the capillaries surrounding the tubule. By filtration, reabsorption, and tubular secretion, the nephridia perform their excretory function and maintain salt and water balance. If time permits, examine a nephridium of Lumbricus under the compound scope. If you are lucky, you will be able to see the ciliary currents moving fluid through the system. Before leaving your dissection of Lumbricus, remove the gut to examine the reproductive structures. Segments 9-13 carry the trilobed seminal vesicles where sperm develop. Observation of this tissue under the compound scope should reveal developing sperm. Sperm are produced in the small paired testes located in segments 10 and 11. Sperm exits the male system via vas deferens which open to the outside in segment 15. The female reproductive orgas will initially be obscured by the seminal vesicles. Below them, in segment 13 you should be able to find two small ovaries attached to the anterior septum. The ovarian funnel projects forward from the posterior septum of segment 13 and leads to a short oviduct in segment 14 which leads to the female genital pore. Segments 9 and 10 contain yellow sperm receptacles (spermathecae), which store sperm from another worm. Examine a prepared cross and longitudinal sections of Lumbricus and identify the internal structures and pay attention to the musculature. CLASS POLYCHAETA Examine the external characteristics of Nereis or any other errant polychaete available in the laboratory. The most obvious difference between Nereis and Lumbricus is the presence of lateral segmental projections in Nereis. These parapodia are used in swimming and crawling movements. Closely examine the parapodium of a Nereis you have anesthetized by placing in a dilute solution of Magnesium chloride and then rinsed.
Each parapodium is biramous, consisting of a bilobed dorsal notopodium and a ventral neuropodium, which functions in both respiration and locomotion. From each there arises a tactic cirrus and a bundle of jointed chitinous setae. They are fine bristles composed of a long blade jointed to a sturdy shaft. The setae arise from invaginated sacs in the parapodia, and each one is secreted by a single cell at the base of each sac. The parapodia are supported internally by two modified setse called acicula. Examine the number and arrangement of the setae. How do they differ from Lumbricus? The anterior end of Nereis shows conspicuous cephalization and differs distinctly from that of Lumbricus in that it bears a pair of prostomial tentacles, paired palps and eyes. The peristomium is also more elaborate in having tentacular cirri. The peristomium is thought to be composed of two fused segments and the peristomial cirri are the modified cirri of normal segments. The mouth is equipped with an eversible pharynx armed with serrated teeth. The pharynx is everted during feeding in predaceous polychaetes such as Nereis.
Position the worm ventral side down in a long, narrow dissecting pan and place the anterior end of the worm in the approximate center of the pan. Most of your attention will be focused on this part of the worm. Before you pin the worm to the wax, be sure its head is at one end of the pan where it can be viewed with the dissecting microscope. When you are satisfied with the position of the worm, temporarily anchor it to and extend the anterior end of the worm toward one end of the pan from this anchor. Make sure it does not extend beyond the end of the pan. Relocate the worm and pins if it does. Pinch the dorsolateral body wall of segment 25 (or thereabouts) with your fine forceps (a little to the side of the midline) to make a fold of the body wall. Use fine scissors to cut through the fold but be careful to avoid cutting across the midline and dorsal blood vessel. Make the cut entirely through the body wall. Insert the tip of the scissors into the opening and cut anteriorly through the body wall close to, but not on, the midline. This will open the coelom from which coelomic fluid will escape. As you begin making the cut anteriorly extend the longitudinal incision from segment 25 to the posterior edge of the prostomium. This incision should lie beside the dorsal blood vessel but should not cross the midline or cut that vessel. This is especially important if you are dissecting a living animal. In Nereis this vessel adheres to the body wall (rather than to the gut wall as it does in Lumbricus) and it is best to leave it attached to the body wall. This means, of course, that you will have to cut its connections with the gut and viscera as you open the animal. Note the connections of the blood vessels before you cut them.
As you cut anteriorly, you will notice that diaphanous transverse partitions, the septa, separate the coelomic space of each segment from that of adjacent segment. Ventral to the gut the septa are incomplete and they do not form complete bulkheads between segments. Under magnification use fine scissors to cut the septa as you make the longitudinal incision. After cutting them, reflect the lateral body walls and pin them (the walls) flat to the wax of the dissecting pan. When you reach the vicinity of segment 10, you will notice large muscles running from the gut tube to the dorsal body wall. These are the pharynx retractor muscles (but they do not originate on the pharynx). Try to leave them intact for the present. Associated with the muscular pharynx are the chitinous jaws that are identifying characteristics of the different polychaetes. There are a variety of lifestyles for polychaete worms: 1. Crawling polychaetes - The crawling polychaetes are basically surface dwellers, living in rock and coral crevices or beneath rocks and shells. In these worms the prostomium is equipped with eyes and other sensory organs, the parapodia are well developed, and the body segments are generally similar. Nereis is a good example of this type of polychaete. In crawling species, the longitudinal muscle layer is better developed than the circular layer, which may even be absent, and the septa tend to be incomplete. Movement is brought about by the combined action of the parapodia, the body wall musculature, and to some extent the coelomic fluid. The pattern of locomotion in Nereis is typical of many crawling polychaetes. The slow crawling movement of Nereis results entirely from the action of parapodia. Parapodial movement involves a backward effective stroke in which the parapodia are in contact with the substratum and a forward recovery stroke in which the parapodia are lifted from the ground. Each parapodium describes an ellipse as it completes one of these two-stroke cycles. The acicula and setae
of the parapodia are extended during the effective stroke and retracted for the forward recovery stroke. The combined effective sweeps of the numerous parapodia propel the worm forward. The right and left parapodia of each segment work alternately rather than simultaneously. In other words, when a particular group of parapodia on one side of the body is executing an effective stroke, the parapodia on the opposite side are executing the recovery stroke. The parapodial action takes place in waves along the side of the worm, with every fourth to eight parapodium (depending on the organism) being in the same phase of the cycle. The waves of activation of the parapodia move from posterior to anterior. In some crawling polychaetes, S-shaped body undulations, in addition to parapodial action, are responsible for locomotion. Body undulations are produced by waves of contraction in the longitudinal muscles of the body wall. These waves of contraction coincide with the alternating waves of parapodial activity just described. The longitudinal muscles on one side of each segment contract when the parapodium on that side of the segment is moved forward; the muscles then relax as the parapodium sweeps backward in its effective stroke.the principal force of propulsion in this type of movement comes not from parapodial movement, but rather from body contractions pulling against points of contact made by the parapodia. An example is Aphrodite aculeate, the sea mouse. Where does this specimen receive its name from? Why? Unlike most other errant (mobile) polychaetes, Aphrodite is unusual in its shape. Compare Aphrodite aculeata with Nereis virens and describe these differences. 2. Pelagic polychaetes - Six families of polychaetes contain exclusively planktonic or pelagic species. They are morphologically similar to crawling forms, except that most of them tend to be transparent and many of them lack setae. (Why to you suppose this is so?) The swimming movements of these polychaetes are S-shaped undulations similar to those described in the preceding paragraph. We have no polychaetes of this type in the lab. 3. Burrowing polychaetes - Many polychaetes, such as Glycera, have become adapted for burrowing. Most of them construct a system of mucus-lined galeries within which they move about. The adaptations of many of these burrowers remarkably parallel those of the earthworms of the class Oligochaeta. The prostomium is reduced and pointed; eyes, palps and antennae are usually absent; parapodia tend to be smaller than those of crawling surface dwellers. Burrowing polychaetes move through the substratum by means of peristaltic contractions. The circular muscle layer of the body is well developed and the septa effectively compartment the coelomic fluid and localize its function as a hydrostatic skeleton. Many polychaetes occupy more or less fixed simple burrows excavated in the substratum and are referred to as sedentary burrowers. Movement through the burrows results from peristaltic contractions. Thus the parapodia are greatly reduced and are in part represented by transverse ridges provided with setae modified into hooks. The prostomial sensory appendages are generally absent in this type of worm, but the head may carry specialized feeding structures. The terebellid worm (family of spaghetti worms due to spaghetti-like tentacles associated with the head), Amphitrite is a good representative of the sedentary burrowers that do not create permanent tubes. What are distinct features of Amphitrite? How does this animal move and how does it feed? 4. Tube-dwelling polychaetes - A tube-dwelling habit has evolved in many families of polychaetes. The tube may serve the worm as a protective retreat or as a lair for catching passing prey. It may provide access to clean oxygenated water above a muddy or sandy
bottom or may permit a worm to inhabit hard, bare surfaces such as rock, shell or coral. One group of tube-dwelling polychaetes contains worms typically carnivorous feeders and extend from the opening of the tube to seize passing prey.they are not greatly different from the surface-dwelling polychaetes in terms of morphology. Prostomial sensory appendages are well developed and the parapodia, which are used in crawling through the tube, are not markedly reduced. Diopatra is an intertidal worm representative of the carnivorous tube-dwellers. Diopatra build heavy, membranous tubes from secretions of special glands located on the ventral surface of the segments. The chimneys of the tubes, which project above the sand or mud sediment, bend and flare at the end like a funnel. These chimneys are covered with bits of shell, seaweed and other debris that the worm collects and places in position with its jaws. Another interesting tube dweller is Pectinaria. This worm constructs a tube by cementing together sand grains. The tube is conical, with the smaller end of the tube opening at the surface of the sand or mud sediment. The head of the worm bears rows of large conspicuous setae that are used in digging. Pectinaria never comes completely out of its tube, but it can move around in the environment carrying the tube with it. Most tube-dwelling polychaetes, however, are highly modified for a sedentary tube-dwelling existence and exhibit characteristics similar to the more sedentary burrowers. Prostomial sensory appendages are reduced or absent, while special anterior feeding structures are often present. The worms usually move within the tube by peristaltic contractions, and the parapodia are reduced and provided with hook-like setae. The body segments are commonly differentiated into regions. Sabella and Hydroides are good examples of sedentary tube-dwelling polychaetes. Sabella and Hydroides belong to a group of polychaetes commonly referred to as feather duster worms or fan worms. In these worms the prostomium has developed to form a spiral or funnel-shaped crown consisting of a variable number of pinnate processes called radioles. When the radioles are extended they look like a feather duster, hence the name. The radioles are rolled up or closed together when the worm withdraws its anterior end into the tube. Sabella constructs a tube of sand grains imbedded in mucus. The worm sorts detritus collected by the ciliated radioles, and sand grains of suitable size for tube construction are stored in two sacs below the mouth. The walls of these sacs produce mucus, which is mixed with the sand particles. A rope-like string of mucus and sand grains is then released when the worm makes additions onto the end of the tube. Hydroides secretes a calcareous tube which is attached to rocks or shells. Two large calcium carbonate secreting glands are located on the prostomium. The dorsal radiole of this fan worm is modified into a long stalked knob called an operculum which acts as a protective plug at the end of the tube when the crown is withdrawn. If Hydroides is available in the lab, be sure to see the quick retraction response it has to disturbance. This rapid response is possible because of a giant fiber nerve system and well developed longitudinal muscles. CLASS HIRUDINEA Examine the external anatomy of the live leeches available in the laboratory (probably Hirudo). Can you locate any setae? In what plane is the body flattened? All leeches have a constant number of segments (34), but secondary external annulation obscures the true segmentation which is apparent in the arrangement of the internal structures. How many annuli are there per segment in Hirudo? One annulus of each segment is marked by a row
of sensory papillae. Five pairs of dorsal papillae in the first five segments are modified into eyes. The more pointed anterior end of the leech bears a small ventral sucke formed from the prostomium and peristomium. The posterior end bears a large ventral sucker made up of segments 25-34. The single opening of the vas deferens is located midventrally at the second annulus of segment 10. The penis issues from this pore. The vagina opens midventrally at the second annulus of segment 11. Like the oligochaetes, leeches are hermaphroditic and cross copulation is the rule. Nephridiopores open lateroventrally at the last annulus of each segment from 6 to 22, but are difficult to see. The clitellum develops seasonally on segments 9-11 that bear abundant secretory cells. The surface arrangement of the musculature in the leech is similar to the other annelids with an outer circular layer of muscles and an inner longitudinal layer. Circular muscles contract with the posterior sucker attached, longitudinal muscles with anterior sucker attached and when the anterior sucker attaches the posterior sucker detaches (movement considered as galloping ). How does the animal swim? Describe the process compared to the attachement? Leeches, also have dorsoventral muscles which maintain the compressed shape and aid in locomotion. The coelomic space is reduced by mesenchymal tissue. This tissue consists of fine capillaries of the coelomic blood sinus system surrounded by globular cells forming a connected system of sinuses. The sinus system is continuous between segments and septa are reduced or absent. The gut is provided with branches called caeca, which increase the surface area for digestion. The reduction of the coelom and lack of a discrete segmental hydrostatic skeleton is reflected in the method of leech locomotion. Locomotion in leeches is drastically different from other annelids. Septa are absent, and the entire body functions as a unit. The coelomic cavity is reduced so does not have as important a role as a hydrostatic skeleton. Movement in leeches is caused primarily by opposing contractions of the circular and longitudinal muscles. The oblique muscles run as a helical spiral along the length of the body. When the body is long and thin the spiral fibers reinforce the longitudinal muscles; when the body is short and thick, they reinforce the circular muscles that produce elongation. When the spiral muscles are neither lengthened or shortened, they maintain internal pressure and enable the leech to lift its anterior or posterior end off the substrate. Prepare a preserved leech for dissection and pin the specimen to a dissecting pan and make a dorsal incision as you did with Lumbricus. Carefully separate the skin from the body and pin it down. Examine the internal structures under a dissecting scope. This is not easy due to the large amount of mesenchymal tissue and the oblique muscles. These tissues can be carefully removed to see internal structure. In any case, note the mesenchymal tissue, oblique muscles, and the lack of a large coelom and segmental septa. How does this compare to the internal anatomy of Lumbricus and Nereis?
COMPARISON OF ANNELID CLASSES Polychaeta Oligochaeta Hirudinea Representative: Nereis virens Lumbricus terrestris leeches 1. Cephalization often elaborate sensory reduced reduced feeding structures 2. Parapodia and/ or setae parapodia often well- no parapodia; a few no parapodia developed with many setae no setae setae 3. Sexuality usually separate sexes hermaphroditic hermaphroditic 4. Reproductive investment many small eggs few large eggs few large eggs 5. Fertilization external within a cocoon internal 6. Development external within a cocoon within a cocoon 7. Hatching Stage free-swimming small adult small adult trochophore larva 8. Habitat chiefly marine fresh water or moist fresh water or soil moist soil 9. Feeding mode Diverse: predators, direct deposit feeders predators or filter-feeders, direct or blood-sucking indirect deposit feeders, ectoparasites herbivores, scavengers