A dissection of the trochid gastropod Tegula funebralis.

Intro: The basic molluscan body plan has been modified through the course of the phylum’s evolutionary history to address the challenges associated with a variety of diverse habitats and life histories. Today we will compare how this basic invertebrate body plan has been modified to suit a benthic, creeping, herbivorous lifestyle representative of many marine snails. We will focus on four major areas: 1) general morphology and orientation of the external body and mantle cavity, 2) cephalization with respect to sensory structures, 3) the circulatory/respiratory system and 4) the digestive system. Throughout the entire lab exercise, you should consider how the basic mollucscan body plan consisting of a shell, muscular/creeping foot, mantle with the underlying visceral mass and associated mantle cavity, and radula have been modified to address the unique demands of this organism’s habitat and lifestyle. Sounds like prime material for annotation to me!

Obtaining your animal: We will be working with live (and anesthetized) snails. There will be some un-narced snails available for live observations but you should use the MgCl2 narced snails for dissection. Your lab instructor will direct you to where you can obtain a specimen.

Sketch 1& 2- External observations: In this section you should familiarize yourself with the general external anatomy of the organism. Make sure you can identify all of the relevant axes (dorsal/ventral, anterior/posterior etc.) Locate and identify all of the obvious features, especially those that reveal the organism’s molluscan heritage such as mantle, foot, shell, visceral mass, radula (note these may not all be apparent in an external view)

Observe first a live and locomoting Tegula in seawater. Note the cephalic tentacles and epipodial tentacles (What do they do?). Apologize to your animal and remove its shell (using a narced specimen). This can be difficult. Tegula's soft body and very hard shell make its extraction akin to cracking open a Brazil nut and removing the meat intact. The best method is to use a vise or screw-clamp. A few minutes of steady stress in the vise's jaw will crack the shell with no damage to the animal. Remember that the animal is attached to the shell at a point along the columella just inside the aperture. The muscle here must be cut for removal. If you end up with what looks like a lump of mutilated flesh, that is exactly what you have and you should go get another animal. All of the features described below should be visible. Many organs are visible at this point, so it is good time to get oriented. Place the animal on its right side (so that the coiling is underneath), so you are looking at its left side. Unless that animal was fully relaxed, the foot will be semi-retracted and look funny and not like Figure 1.

Figure1: External view of T. funebralis (shell removed). Top – left lateral view. Bottom – right lateral view

Three things are immediately obvious: the green or white gonad making up most of the upper spiral, the brown digestive gland, and the small spiral caecum between them (see Figure 1a). Anterior to the digestive gland is the pericardium and the orange Gland of Nelia. Arising from this area and traveling forward toward the head is the bipectinate ctenidium, visible through the mantle tissue as a long yellowish structure. Turn the animal over on its left side (so you are looking at its right side) and three more organs are visible (see Figure 1b): the long darkish rectum, tracing a path analogous to the ctenidium's: the left kidney, lying on top of and partially enclosing the intestine: and the hypobranchial gland, attached to the mantle tissue above the rectum and visible as orangish blotches. The pericardium and gland of Nelia are visible on this side as well and can be seen between the intestine loop and the gonad. If you can't make out the kidney or hypobranchial gland, don't worry, as they will become more visible when the next step, entering the mantle cavity, is performed.

Sketch 3 - The mantle cavity:After you view the external anatomy, the animal should be pinned down so that its dorsal surface faces up. Now make a cut from the edge of the mantle toward the base of the visceral hump. It is best to cut the mantle tissue midway between the two cephalic tentacles. Be careful not to sever organs directly beneath. The cut should extend as far back as the cavity does (to the pericardium but no further). Figure 2: Exposed mantle cavity of Tegula with mantle cut from anterior edge to just anterior of the pericardium. (Dorsal view – anterior at the top)

When the tissue flaps are pulled to the side the most prominent organ is the ctenidium,

lying to the left of the cut. It is orange, feather-like, and relatively long (can you elucidate the direction and route of water flow through the mantle cavity, based on the ctenidium's shape and position?). Next to the tip of the ctenidium at or near the edge of the mantle is the small osphradium. It too is orange but only a few millimeters in diameter.

On the wall of the mantle around the rectum is a fine network of tubules. This is the hypobranchial gland (see Figure 2). It may be partially blocked from view by the kidneys and the rectum. The kidneys themselves partially enclose the rectal intestine just posterior to the hypobranchial gland. It is often difficult to distinguish the right kidney from the left. The best way is to find their at their anterior ends (this is done in the next dissection).

The distal portion of the rectum appears to come out of the kidneys. If you lift up the kidneys it is possible to trace the intestine back up to the gland of Nelia, where it disappears.

The pericardium lies pretty much on top of the bend of the intestine and just anterior to the gland of Nelia. The efferent vessel of the ctenidium leads directly into it. In a live animal, the heart's auricles and ventricle may be beating inside the pericardium.

The large buccal mass contains the radular sac, which you can see outlined very faintly in this prep now. Alternative cut for the mantle cavity dissection.:

The methods described above for accessing the mantle cavity requires that you cut through the afferent blood vessel (which enters the ctenidium about midway along its length) in order to expose the kidney and intestine. It is possible to expose the mantle cavity without cutting the afferent blood vessel, but this requires that your incision be at the extreme left end of the mantle cavity and run up to just before the pericardium (Fig. 2B). If done properly, this will allow you to peel back the entire flap to the mantle tissue to expose all of the organs intact (but backwards in orientation). You may wish to try this approach if you are seeking a challenge, but the standard approach is your safest bet.

Fig. 2B: Alternative cut to expose the mantle cavity (Dorsal view – anterior at the top)

Sketch 4 - Circulatory and respiratory systems: As a result of exposing the mantle cavity along its central axis, the ctenidium will be on the left, the rectum and kidney on the right. This usually results in cutting the afferent branchial vessel, (When a vessel is punctured, it leaks milky-blue fluid, an oxygenated hemocyanin.) If your original cut hasn’t already reached it, cut back to the pericardial sac, being careful not to cut into the efferent branchial vessel. Trace the course of the blood: Starting from the visceral sinus, go up the back of the right nephridium and through it to the afferent (and most likely severed) branchial vessel, which also drains the hypobranchial gland; then continue to the other side, through the pericardial wall to the atrium, to the ventricle, then out the aorta to the posterior and cephalic arteries, which emerge from under the right. In all likelihood you will not be able to distinguish the latter vessels, but you should be able to locate both branchial blood vessels (afferent and efferent) the left atrium, and the ventricle (especially when you remove the pericardial tissue). Last of all, cut and pull away the tissue covering the pericardial cavity (carefully) (Figure Figure 3). Note the two atria will be apparent. The left will be a more distinct triangular flap between the intestine and the wall of the pericardium, while the right (a vestigial organ –why?) will be on the opposite side and towards the rectum. The ventricle may or may not be still pumping, and is virtually indistinguishable from the intestine it surrounds. It is clear in color and has a wrinkled, rubbery texture. Remember molluscs (including snails) typically have an "open" circulatory system. Open circulatory systems still have lots of vessels but they lack capillaries and other such connections between their arterial and venous elements.

Photographs of the exposed mantle cavity (left – dorsal view anterior to the left) and the exposed pericardium (right – dorsal view, anterior to the left ): Fig 3: Location of pericardium relative to kidney and Gland of Nelia. Top left: right lateral view (anterior on the right). Bottom right: dorsal view (anterior at bottom)

Sketch 5- The digestive tract

Returning to the main section of the mantle cavity, follow the intestine tract up the right side to locate the kidneys. The left kidney is a whitish convoluted structure encircling the intestine. Its nephridial pore lies at the anterior end of the organ. To the left and slightly above this opening, the right (yes) kidney's smaller pore can be found. Directly above the kidneys, at the termination of the intestine, lies the anus. It is in this region of the mantle cavity that you'll find the conspicuous orange-yellow hypobranchial gland, which secretes mucous for the formation of fecal pellets. The right salivary gland is situated behind the buccal region, often under the left side of the kidneys. The left salivary gland is located in the corresponding area on the left side of the animal. These glands secrete enzymes involved in the digestion of carbohydrates (excluding cellulose). Having accomplished these feats, you can now return to the gland of Nelia.

The large brown digestive gland extends posterior from the gland of Nelia to the "tail' end. you'll see an interestingly coiled structure located slightly to the left of the midline and ahead of the gonad. This is the spiral caecum. You may wish to tease away the digestive gland and follow the coil anterior to the point at which it connects to the stomach and remove some of the digestive gland to expose the dorsal surface of the stomach but this can be messy so you may opt to give it a pass. The gland of Nelia extends anterior along the esophagus tube. According to Libbie Hyman (The Inverts: Mollusca I), there is some research indicating that this gland secretes cellulases utilized in the digestion of cellulose. The pharynx, ahead of the esophagus, contains radular cartilage surrounded by pinkish striated musculature. The radular cartilage is used in conjunction with the radula to grind Tegula's food. Now open the buccal cavity and extract the (surprisingly delicate) radula. Get it all, way back into the radular sac, and note how the radula rides on a muscular lump, the odontophore. As the radula is worn out, it is absorbed in the anterior and renewed in the posterior: a neat morphological trick! Figure out how the radula licks and scrapes up food for the snail. You may wish to remove the radula and mount it for close scrutiny under the compound scope. Some clever epi-illumination will help reveal its intricate array of teeth, which you should be sure to see.

Figure 4: Dorsal view of the digestive tract of T. funebralis (portions of the digestive gland have been removed).

By now you should be completely awestruck by the incredible elegance of the body design of this seemingly simple snail. I guess the advertising geniuses in Madison Avenue were right when they said it’s all in the packaging. \ Phylum: Mollusca Class: Gastropoda Sketch # Title Est. time Notes 1 Ext. anatomy/Live observation 20 min Label view clearly LABEL: shell, foot, operculum, head, cephalic tentacles, epipodial tentacles 2 & 3 General internal anatomy 45-60 min LABEL Left: gonads (male or (L and R sides) female?), digestive gland, spiral caecum, gland of Nelia, pericardium, ctenidium, mantle, columellar muscle attachment (CMA), neck lobe LABEL Right: rectum, left kidney, intestine, hypobranchial gland 4 Mantle cavity 30 min LABEL: foot, osphradium, hypobranchial gland (HBG), radular sac, ctenidium efferent vessel, gland of Nelia, digestive gland, spiral caecum, buccal mass, rectum, left/right kidney, pericardial sac, gonad 5 Circulatory system 30 min Must use alternative mantle cavity dissection if you want to see fully intact circulatory system NOTE blood flow (see below), focus on organ systems involved and outline other organ systems for reference LABEL: pericardium, left atrium, ventricle, afferent/efferent vessels, ctenidia 6 Digestive system 30 min Note digestive flow LABEL: esophagus, intestine, gland of Nelia, digestive gland, spiral caecum, stomach, left/right kidney, HBG, anus 7 Radula 20 min Radula, odontophore, radular sac

Conclusion: Relate the form and function of this organism to the habitat and environment in which it lives. Discuss the similarities and differences between Tegula and the ancestral mollusc/gastropod form.