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PHYLUM MOLLUSCA Introduction Because the Mollusca are designated as a phylum we know that all the organisms in the taxon must share a common body plan that they have inherited from the ancestral mollusc; it’s the very definition of a phylum after all. But, if we were to put a representative from each of the different classes, both extinct and extant, on the lab bench we would have a tough time trying to find the characteristics of the ancestral body plan that unifies the classes of the phylum. That’s because each class is a spectacular examples of invertebrate adaptive radiation with the result that seemingly unrelated animals, such as snails, slugs, clams, the octopus and squids are all related to each other and share a single common ancestor. Whatever it looked like the ancestral Mollusc’s body plan was sufficiently flexible that it allowed its descendents to adapt with a range of behaviors, and habitats. From sedentary filter feeders to rapid swimming active predators, and just about everything in between, there is a mollusc that does it. The adaptive potential of the ancestral molluscs is also seen in where you find molluscs; freshwater, marine and terrestrial environments. Their general appearance wouldn’t necessarily give us any obvious clues to why these animals are all included in one phylum but there are a variety of traits, or characters, that unify the group and you should look for these in each of the specimens. These include the presence of a muscular foot, involved in locomotion, and a sensory head that was also associated with food acquisition by the unique molluscan feature of a radula. Another is the visceral mass, which is dependent on cilia for its function, and positioned dorsally surrounded by the protective shell. The shell is secreted by the underlying mantle extending out from the body creating a mantle cavity inside which you’ll find the ctenidia, the principle respiratory structures of molluscs. Polyplacophora These animals are referred to as chitons and their body form is specially adapted for the rough conditions associated with the intertidal zone of the oceans. When chitons are active they slowly creep across the rocks feeding on encrusted algae and other organic debris. If threatened they can roll up into a ball surrounded by the protective armor of their shell. Fig. 1. External features on the dorsal surface of a chiton. © BIODIDAC Phylum Mollusca -1- Digital Zoology LabManual © Houseman External anatomy The most obvious external feature of these molluscs is the set of eight,overlapping shells, or valves, on dorsal surface covering and protecting the visceral mass underneath. The posterior edge of each shell overlaps the front of the shell behind it. As a result, the shells are articulated, and can move relative to each other allowing the chiton to cling tightly to the rocky surfaces on which it crawls or curl up in an armored ball when attacked. Around the edge of the chiton is a muscular girdle with lateral edges of the eight valves embedded in it. The girdle is an exposed part of the mantle, the rest is underneath the plates and, depending on the specimen,you will be able to see the needle-like calcareous spicules embedded there. The most primitive molluscs didn’t have a shell and were protected by spicules much like those around the edge of the chiton. It’s believed that when a secreted shell did appear it took the form of plates like those seen in the Polyplacophora. Later modifications of the shell solidified it in a single structure consisting of only one valve, univalve, that could be wound or folded in a variety of ways. Ultimately the shell disappears in some molluscs. Fig. 2. Major anatomical features on the ventral surface of a chiton. © BIODIDAC The large oval foot dominates the ventral surface of a chiton and along its lateral edges are the mantle cavity includes grooves formed from a trough between the foot on the inside and the fleshy girdle. Inside the mantle cavity you can see the multiple ctenidia used for gas exchange. The mouth is easy to see at the anterior end but there are neither eyes nor tentacles associated with it. At the opposite end, the anus is located on the roof of the mantle cavity, on the tip of a small papillae. Like all molluscs cilia on the surface of the ctendia propel water through the mantle cavity pulling it in at the anterior end surrounding the mouth, down the two mantle cavities on each side, and over the ctenidia. At the back the left and right mantle cavities fuse to form a single exhalent canal where the anal opening is located. If you look closely in the region of the last few ctenidia you may also be able to see nephridiopores or gonopores that open into this posterior part of the mantle cavity. Gastropoda Gastropod shells One gastropod trait that is easy to recognize is their spiralled shell; the most effective way to package the increasing size of the visceral mass as the snail grows. The shell consists of only one piece, a univalve, molded into a cone wound on itself. Essentially the snail winds its shell up and lives underneath it. A snail’s shell is a protective safe haven where it can pull in its whole Phylum Mollusca -2- Digital Zoology Lab Manual © Houseman body when threatened. Some snails have an extra piece of shell on the dorsal surface of the foot, an operculum, that closes to door behind them. Overwintering snails secrete a thin layer of calcified slime across the opening. Fig. 3. Major structures of a gastropod shell. © BIODIDAC Examine the intact and sectioned conch shells to understand the spiralled nature of the shell. The main parts of the body includes the head and the foot extending out of the opening of the shell, the aperture. Not all apertures open in the same direction. Place the apex of the shell away from you. Is the aperture’s opening on the left or right? If it’s on the right is dextral, on the left sinistral just like right and left-handedness. One complete circle of the shell is a whorl and the edges of each whorl are connected to the next by suture lines. These lines are often sculptured and can form spines. Like all mollusc shells, growth lines are visible on the surface of the shell and the oldest part of the shell is the apex. In the shells that have been cut open identify the columella. Are there individual chambers in the shell, or is it one single continuous chamber? Not all gastropods have shells; slugs have discarded the cumbersome shell. Helix Helix is a terrestrial herbivore that prefers to feed at night, or after a rain when conditions are damp. Many gardeners battle the nocturnal dining of snails and slugs in their gardens. They overwinter by crawling under rocks or digging into the soil. External Anatomy Take a relaxed specimen; its body extends outside of the shell. Locate the apex of the shell and the anterior and posterior ends of the animal. Only the head and foot extend from the shell, the visceral mass remains protected inside. The mantle’s edge is thickest in the region of the collar. The pneustome, the opening to the mantle cavity, is located on the surface of the collar. One of the best ways to find the pneustome in preserved specimens is to gently squeeze the body of the snail and watch the collar for where preservative comes out. These are terrestrial animals and the mantle cavity has been modified as a lung that we will see later when we remove the shell. The anterior position of the opening to the mantle cavity is a consequence of torsion, another is the anal opening positioned in front of the animal instead of behind. Locate the anus just beneath the pneustome, and the mouth with its three lips. All three openings are located on the anterior part of the body, and when you see this in a mollusc you know you have a gastropod. Where are the openings to the anus and mantle cavity usually located in molluscs? The Phylum Mollusca -3- Digital Zoology LabManual © Houseman gastropods assymetric body plan is best seen by obersving the position of the anus and opening to the mantle cavity on the side and the displacement of the shell to the side of the foot, rather the above. Below the mouth is the opening to the pedal slime gland that lays down the mucus trail used in locomotion. There are two pairs of retractable tentacles on the head the most anterior are shorter than the longer posterior pair with an eye at the tip. The opening to the reproductive system, the genital pore, is below and just behind the anterior tentacle on the right side of the head. To see it you may want to let the surface of the snail dry off a little. To see the remaining external features you will have to remove the specimen from the shell. This is not an easy task because the snail’s body is wound right up through the shell to the very apex. Use a hammer to crush the shell by gently hitting the delicate apex of the shell. Be careful when you do this, too much force and the sharp edges of the broken shell will damage the specimen. Just enough force and the shell will break and you will be able to remove it to expose the underlying snail. The whole body is covered by the delicate mantle that thickens only at the collar. This thicker part of the mantle results in collar shell being laid down faster compared rest of the mantle edge; this is what creates the spiral of the shell. The dorsal surface of the front half of the mantle cavity well vascularized and you will see the blood vessels that carry the haemolymph into the mantle for oxygenation. How is air pumped in and out of the mantle cavity? The single metanephridium, kidney, is dark and can be seen in the posterior half of the mantle cavity occupying the second half of the first visceral whorl. Why are the excretory structures not paired? Below the junction of the metanephridium and the lung is the heart surrounded by the pericardial cavity. The heart consists of two chambers, paired auricles, or atria, receiving the blood from the lung and a single ventricle pumping blood from the heart to the rest of the body. On the inner edge of the mantle is the rectum. If you were unable to locate the anus in your previous observations cut a small opening in the rectum and use a blunt probe to find the exterior anal opening. The digestive gland and reproductive system fills the remaining whorls of the visceral mass. Near the apex of the coil you will be able to see the albumen gland and in some cases the ducts leading to the ovotestis found at the apex. Internal Anatomy The dissection of the snail is not one of the easiest but if you are careful, and patient, you will see all of the structures. What makes this dissection so hard is that the spiral of the visceral mass means that the structures that you need to see are wound around inside the spiral as well. Fig. 4. Major anatomical features of the snail. © BIODIDAC Circulatory and excretory system Submerse your specimen under water and to one side of the dissecting dish so that you will be able to look at it under the dissecting scope. Use a pair of fine scissors and cut through the collar in the region of the pneustomes. Cut along the edge of the collar towards the rectum and then along the length of the mantle on the inner edge of the whorl. Pin aside the roof of the mantle cavity and identify the blood vessels; efferent blood Phylum Mollusca -4- Digital Zoology Lab Manual © Houseman vessel on the midline of the cavity’s roof is the easiest to see and it carries blood to heart. Open the pericardial cavity to expose the large atrium and smaller, muscular ventricle. The ventricle pumps blood into an aorta with two main branches; the visceral aorta supplies blood to the visceral mass and the cephalic aorta that supplies the head. The metanephridia is also located on the roof of the mantle cavity and on the piece of mantle you have just exposed. Locate the metanephridia and the duct that drains it. Digestive system Cut through the floor of the mantle cavity and collar along the midline. Continue the cut forward between the tentacles until you reach the mouth. Be careful that you don’t cut too deeply and damage any of the underlying structures. Fold the two flaps of tissue back so that you can see the structures underneath. The body cavity that you have exposed is the hemocoel. Working back from your first cut carefully cut along the midline and gently remove the thin skin of mantle that covers the underlying organs. Reproductive structures lie primarily on the right; digestive on the left and of these the pharynx is the easiest place to start your observations. A short esophagus connects the pharynx and the buccal mass to the crop. On either side of the crop are the salivary glands with a duct that empties into the pharynx. The crop is connected to the thin walled stomach located in the second whorl. The paired digestive glands that fill the rest of the visceral whorl connect to the stomach. The intestine leaves the stomach, bends back on itself, and extends towards the anterior before becoming the rectum opening to the outside through the anus. Fig. 5. Major anatomical features of the snail. © BIODIDAC Later, when you have finished your observations of the reproductive system, open up the pharynx to see the radula inside. Below the radula locate the jaw. The radula works against the jaw to grind up the ingested meal. Nervous system The main nerve ring may be apparent over the buccal bulb. The remaining part of the nervous system is difficult to trace as a consequence of torsion and there really isn’t any reason to try and find it. Reproductive system Snails are monoecious and you’ll be able to see most of the structures of the male and female reproductive systems. Be sure to spend a bit of time unwinding things so that you can see the main features. When you cut through the floor of the mantle cavity to expose the digestive system you will have also exposed the different parts of the reproductive system. We will need to find a few landmarks from which we can map the remainder of the system. The mucus glands, with their filamentous components and the dart sac are the easiest to find. As its name suggests the dart sac contains a dart that is jabbed into the side of this snails partner during copulation. Is the dart a part of the male or female reproductive system? Phylum Mollusca -5- Digital Zoology LabManual © Houseman At the base of the dart sac is the junction of a number of tubules from the male and female parts of the reproductive system. The junction of the female is the vagina containing the openings to the seminal receptacle and the hermaphrodite duct. Adjacent to this is the penis, which is in turn connected to the flagellum. The flagellum is wound up on itself but if you gently tease the various tubules apart from each other you will be able to find the tip of the flagellum. Follow it to its base and you will find the penis. Be careful as you expose the flagellum because the delicate sperm duct connects to the flagellum above the penis. Expose the common duct extending from the junction of the male and female reproductive tracts back to the albumen gland. It is easy to identify by the appearance of its two components: a large globular gland of the female system and the tubular sperm duct attached to it. The albumen gland attaches to the common duct and differs from the digestive gland by its shape and lighter color compared to the digestive gland. Extending from the base of the albumen gland is the hermaphroditic duct that leads to the ovotestis which is buried in the tip of the digestive gland. There is only one part of the reproductive system that we have not identified, the seminal receptacle. This is a ball shaped structure, also found in the area of the digestive gland attached by a filamentous duct to the vagina. It is often difficult to see this intact but you should be able to find the seminal receptacle itself, and with a careful dissection its connection to the remainder of the system. Gastropod diversity Examine the different gastropod specimens. How have they adapted to the problems associated with torsion? To answer this question you might want to follow how water moves through the mantle cavity and note the position of the anal or excretory openings. Specimens if available include: limpets, the garden slug, Limax, conch, and nudibranch Scaphopoda Fig. 6. Major anatomical features of a scaphopod. © BIODIDAC There are two plastic mounts that show the internal anatomy of the tusk shelled Scaphopods. These animals have a characteristic tooth-like shell and the mantle cavity runs the length of the shell. Compare these specimens to the diagrams showing the internal anatomy of these animals. Phylum Mollusca -6- Digital Zoology Lab Manual © Houseman When these animals feed, thin tentaclular captacula reach out from the mantle and pick up organic matter on their sticky ends. The food is then passed to the mouth. A U-shaped digestive system opens into the mantle cavity and wastes are passed out through the tip of the tusk. The gonads are located internally more towards the tip of the shell and also release their contents into the excurrent flow. The foot is used to burrow in a manner similar to that seen in the bivalves. Bivalvia Clams and their bivalve relatives are one of only a few mollusc classes found in both freshwater and marine environments. In both places they're specialized as filter feeders, and like most filter feeders they're essentially sessile. To accommodate their way of life, bivalves have made some major modifications to the mollusc body plan. The most obvious is that the body is compressed laterally with the mantle folded down over the sides in two lobe-like sheets. The shell matches the shape of the mantle's two parts. The shell has two valves that cover each side of the animal and an elastic hinge ligament connects the sides across the top. The two valves give the class their name, the bivalvia. But, be careful. These are univalve molluscs because the original single valve is only folded! The lateral compression of the bivalve body not only changes the shape of the shell but also the organs of the visceral mass. Instead of sitting above the muscular foot, the visceral mass is partially buried in the foot. The only part still visible above the foot is the pericardial cavity on the dorsal side. Another modification for the filter-feeding lifestyle has resulted in the loss of the radula and head. Fresh water clam - Unio or Anodonta External Anatomy Identify the dorsal hinge ligament and next to it the paired umbos on the outer surface of the shell. The umbo is the oldest part of the shell and is used to identify the clam’s anterior end. Look closely at the surface of the shell and you’ll see a series of concentric rings extending from the region of the umbo. Just like the rings of a tree, each of the rings on the surface of the shell represent different rates of growth at different times of the year. Look at the animal from the anterior and posterior ends. Where are the excurrent and incurrent openings and how are they positioned relative to each other? Orient the clam so that its anterior end is on your right and the dorsal surface is uppermost. The side facing you is the right-hand side of the clam and we’ll be removing the right shell. To do this, use a scalpel to remove part of the hinge ligament. This will weaken the ligament and make it easier to open the shell. Commercially available preparations usually have a small wooden peg holding the ventral side of the shell open. If not, partially open the shell and beginning at the dorsal posterior region, separate the sheet of tissue, the mantle, from the shell using a blunt Phylum Mollusca -7- Digital Zoology LabManual © Houseman probe. Once the mantle has been separated from the shell reach in over the mantle and with a scalpel and cut the large anteriorly and posteriorly located muscles. Carefully lift the shell off and as you do, separate the tissue that remains attached to the shell. Fig. 7. Features on the inner surface of a freshwater clam shell. © BIODIDAC Examine the inner surface of the shell you removed. Near each end are the large adductor muscle scars. These muscles run from the left to right sides connecting the two valves to each other. Contraction of the shell adductor muscles closes the shell and the hinge teeth insure that the two shells align properly as it closes. How does a clam open its shell? Next to each of the adductor muscle scars is a scar from the attachment of the foot retractor muscle. The anterior muscle scar lies a bit above and behind the anterior adductor. The posterior retractor lays next the posterior shell adductor. As their name implies the foot retractor mucles pull the foot into the shell. How does a clam extend its foot? The pallial line extends from the adductor muscle scars and runs along the edge of the shell. This scar is created by the attachment of muscle along the mantle edge. Fig. 8. Major structures inside the mantle cavity of a bivalve. © BIODIDAC Break off a piece of shell from the valve you have removed and identify its three layers. These include an inner mother of pearl layer, or nacreous layer; a middle prismatic layer and the outer periostracum. Internal Anatomy If it isn’t already, fold the mantle back over the body of the clam lying inside the left shell. The mantle is fused to the dorsal surface of the animal and on the ventral side it hangs free and encloses the mantle cavity. With the mantle still covering the body look at the posterior edge. The incurrent and excurrent openings are only visible when the two sides of the mantle lie next Phylum Mollusca -8- Digital Zoology Lab Manual © Houseman to each other. The incurrent opening has a more ragged edge than the excurrent opening and this helps to filter the incurrent water. In many bivalves the two sides of the mantle fuse and are extended as siphons. Lift the edge of the mantle and remove it by cutting along where it fuses to the dorsal surface of the body. The two most prominent structures visible underneath are the ctenidia and the muscular foot. Respiratory system W-shaped ctenidia are found on both sides of the foot and are covered with cilia that pump water into the mantle cavity and across the gill surface. The ctenidia are attached to the visceral mass along their dorsal surface with their ventral edges suspended inside the mantle cavity. Each ctenidium consists of two demibranchs each formed from the fusion of two lamella. The lamella are fused at their distal edge and the space between them is divided into a series of water tubes running from the bottom to the top of the demibranchs where they empty into the suprabranchial chamber. Water enters the demibranchs through ostia on the surface and these connect to the water tubes inside. It then moves dorsally in the filament to suprabranchial canals and out the excurrent opening. Fig. 9. Internal anatomy of a bivalve. © BIODIDAC Cut a piece of gill from your specimen and look at its edge under the dissecting microscope to see the paired lamella, the connections between them and the water tubes inside. Examine the side of the same piece under low power to see the grid of ridges and connectives that form the demibranchs. Blood vessels are embedded in the tissue and as the water flows through, the blood is oxygenated. Fig. 10. Detailed structure of a demibranch. © BIODIDAC Phylum Mollusca -9- Digital Zoology LabManual © Houseman In freshwater species the female gills may be enlarged. They are used as brood chambers and small larval glochidia may be contained inside. This does not occur in marine gastropods where the larval stage is free swimming. Prepared slides of glochidia are available. Circulatory system Bivalves have an open circulatory system with a heart lying inside the dorsal, pericardial cavity, underneath the hinge. The largest cavity of the body is the hemocoel and blood from the heart is pumped into the hemocoel where it bathes the various tissues and organs before passing back to the heart through the ctenidia. If it’s not already open, use a fine pair of scissors to open the pericardial cavity by making a cut along the dorsal edge of the cavity. The intestine passes through the pericardial cavity and the three-chambered heart is wrapped around it. Paired thin walled atria, auricles, on either side of the cavity connect the ctenidia to the heart. These triangle structures are often damaged when the pericardial cavity is opened and if you don’t see the atria on the side nearest you, gently move the intestine and look on the opposite side of the pericardial cavity. Blood in the atria passes through large ostia and into the single ventricle. When the ventricle contracts, blood flows into either the anterior or posterior aorta carrying the blood to the hemocoel. Fig. 11. Circulatory system in a bivalve. © BIODIDAC Excretory system The metanephridia are located just underneath the pericardial cavity and in front of the posterior adductor muscle. They appear as dark glandular structures and the opening in the wall of the pericardial cavity is difficult to see. The metanephridium is composed of two parts the glandular part near the heart, and the duct that carries the urine to the nephridiopores opening on the side of the visceral mass near the base of the inner demibranchs. This opening is also hard to see. Fold the gills up and see if you can locate it. How do metabolic wastes pass from the circulatory system to the metanephridia? Digestive system The same water used to aerate the gills also contains particulate food. Cilia on the surface of the gill move the particulate food to the ventral margin of the demibranchs and cilia along the grooved edge pass it forward to the labial palps. There are two pairs of labial palps, inner and outer, and these fuse above the mouth. The labial palps are also covered in cilia and their beating propel food particles into the mouth. We’ve already made a note of the intestine running through the pericardial cavity; follow it towards the anus that empties into the suprabranchial chamber. The rest of the digestive tract is contained in the visceral mass embedded in the foot. To be able to see this you’ll have to carefully remove the muscular tissue of the foot. This is best done by using a sharp scalpel and gradually slicing away tissue starting in the region of the labial palps. The slices should be from dorsal to ventral and parallel to the main axis of the body. With each slice you’ll expose a bit more of the underlying digestive system starting with the mouth and Phylum Mollusca - 10 - Digital Zoology Lab Manual © Houseman short esophagus that leads to the stomach. The greenish tissue above the stomach is the digestive gland. The intestine is embedded throughout the foot and as you continue to remove thin slices of tissue you’ll see sections of the convoluted intestine. Fig. 12. Structure of the glochidial larva of freshwater clams. © BIODIDAC Reproductive system Although the sexes are separate there is no easy way to tell the sex of your specimen because morphologically the gonads are the same in both sexes. If you’re specimen has the modified gill with glochidia inside then you’ve got a female. The tissue of the gonads spreads throughout the foot and is often difficult to distinguish from the spongy musculature of the foot. You’ll need to use your dissection scope and a well-flooded specimen to be able to see the difference between the two. Nervous system The nervous system is difficult to see and in fresh specimens the various ganglia will have a pinkish color. It consists of three pairs of ganglia; cerebral, pedal, and visceral ganglia. The visceral ganglion is located on the surface of the posterior adductor muscle. The brain is located just above the mouth and behind the anterior adductor muscle. Three major nerves radiate from the brain and connect to the visceral ganglion, the pedal ganglion and the mantle, and the pallial nerve. The pedal ganglion is located in the base of the foot Cephalopoda A cephalopod’s visceral mass has been stretched along the dorsoventral axis above the foot, bringing the head and foot closer together on the ventral side. That’s how they got their name, Cephalopoda (head, foot). The mantle surrounds the visceral mass, and ancestrally a hard shell surrounded all of this to form an elongated cone-shaped shell with the head and foot poking out the open end. It was easier to point the tip of the shell in the direction that the animal was moving. Cephalopods swim with what was their original dorsal surface pointing in the direction they move, rather than up. In most animals the surface of the body facing into the direction of movement would be the functionally anterior surface and this is usually the same side of the body where the mouth and head are located. In cephalopods the anterior head and mouth has now become the new dorsal side although the squeezing together of the foot compressed the anterior/posterior axis and the oral opening and head still have a functionally anterior position but its not facing in the direction that they swim. The result of this is that cephalopods swim backwards! With a larger visceral mass on top of a muscular foot, cephalopods were faced with the same problem as gastropods had for finding a way to make a more compact body. They used the same solution, although they wound their shells in a different way. The fossil record includes cephalopods with unwound, wound, and even partially wound shells, and the diversity of fully wound ammonite fossils is good evidence of the successful body plan. At one time these cephalopod predators ruled the ancient oceans, but now only Nautilus remains to give a hint to what these ancient animals looked like. Phylum Mollusca - 11 - Digital Zoology LabManual © Houseman Shelled cephalopods disappeared at about the same time as jawed predatory fishes appeared. It’s possible that the two events are related. The mollusc adaptation to these more active and agile newcomers was another modification to the mollusc body plan to become just as active and agile–the result are the cephalopods we see today. Nautilus shell The only living cephalopod with a shell is Nautilus and even in this animal it differs from that of the other living Molluscs. In Nautilus the pearly shell consists of a series of closed chambers separated from each other by septa and connected by the small openings created by the siphuncle. This animal lives only in the very last chamber and the body does not extend back into the shell. How does this differ, for example, from the spiral shell of a Gastropod? Changes in the ionic composition of the fluid in the siphuncle move fluids in and out of the chambers. This affects the levels of gas in the chambers and has the overall effect of altering the buoyancy of Nautilus. In the other Cephalopods the shell has become reduced and internal. It may be large such as the cuttle bone in cuttle fish or just a thin “pencil” or strengthening rod, or pen, as in the squid Loligo. Fig. 13. Ancestral body surfaces of a squid as seen from the functionally ventral side. © BIODIDAC Loligo Squids in the genus Loligo are found around the world from warm shallow waters to the deep abyssal depths of the oceans where they feed on a variety of small crustaceans and fish not fast enough to escape from these predators. External anatomy To better understand how cephalopods have modified the mollusc body plan it’s important to orient yourself by locating the head, foot and dorsal visceral mass. The squid’s body is divided into two main regions. The first is the elongate, and somewhat conical visceral mass surrounded by the mantle. Below this the head and foot that have fused. The last region includes the arms, and tentacles surrounding the mouth. The mouth is the original anterior part of the body, the funnel the posterior. Nautilus and the ancient cephalopods wound this visceral mass up, modern cephalopods just tip over and swim with the ancestral dorsal surface pointing in the direction that they travel. The result; the anterior side is functionally dorsal, the posterior is functionally ventral, the ventral functionally anterior, and the dorsal surface functionally ventral and as was Phylum Mollusca - 12 - Digital Zoology Lab Manual © Houseman mentioned earlier cephalopods end up swimming backwards. From here on the terms dorsal, ventral, anterior and posterior refer to the functional organization of the animal; trust me it will be less confusing. If you are using a fresh specimen, take a look at the body surface. The small dots of coloration are the elastic capsule chromatophores that squids use to change colors. There are eight arms and two tentacles surrounding a central mouth. Place the dorsal surface uppermost in your tray, if you’re following the functional orientations we just described that means funnel down. The five arms on each side are numbered, starting from the dorsal surface as one to five right, and one to five left. Using this numbering scheme appendages L1 and R1 are the smallest arms and R2, L2, R3, L3, R5 and L5 are larger. Arms have two rows of suckers and are not retractable. This differs from R4 and L4, the tentacles. These have four rows of suckers on an enlarged tip of the tentacle referred to as a peduncle. Unlike the arms, tentacles are retractable being shot out to capture prey and shortened to bring it into the mouth. Fig. 14. Arms and tentacles surrounding the mouth of a squid. © BIODIDAC Take a close look at the suckers of the arms under the microscope. Examine a sucker near the base of an arm; those at the tip are the youngest and as a result much smaller. The cup of the sucker is surrounded by a chitinous ring with a central muscular suction cup. Each sucker is attached by stalk or pedicle. If you have a male, arm L5 will be modified for sperm transfer and is referred to as the hectocotylus. Its suckers are small and located on the end of much longer pedicles. The hectocotylus arm is used in mating and sperm, contained in a spermatophore, are attached to these modified suckers before being passed to the female. In some species the tip of the arm and its package of sperm breaks off. It’s no great loss to the male; in cephalopods damaged arms can be regenerated. Lets turn our attention to the mouth region. Bend back the arms and tentacles attached to each other by a muscular membrane surrounding the central mouth. Inside this is a second membrane, the ruffle-edged peristomial membrane. In female squids the peristomial membrane is modified into a horseshoe shaped seminal receptacle in the middle and below the mouth. Sticking out from the mouth you should be able to see the beak-like chitinous teeth used to tear apart captured prey. We’ll take a closer look at the buccal bulb later in the dissection. A pair of eyes on the head are remarkably similar to mammalian eyes; an excellent example of convergent evolution. What is convergent evolution? Identify the cornea, iris, pupal and lens. How does this eye differ from the mammalian eye? Just behind the eye, and near the base of the arms is the aquiferous pore that stabilizes fluid pressure on the eye as the squid dives. In front is a crest of tissue referred to as the olfactory crests and next to them the olfactory grooves. They are chemosensory and positioned in the incurrent flow of the mantle cavity. The cone shaped funnel, siphon, is located on the ventral surface of the head and water is forced out of the funnel for jet propelled locomotion. Phylum Mollusca - 13 - Digital Zoology LabManual © Houseman The mantle, with two lateral fins that are extensions of the mantle cavity, surrounds the visceral mass of the body. Muscles in the fins allow graceful forward and backwards motion and rapid changes in direction when necessary. The medial mantle artery is visible from outside and is located on the midline of the body between the collar and tip. Look at the edge of the mantle, the collar, and you’ll see three divisions created by three projections around its edge. On either side of the funnel are the pallial cartilages that form cartilaginous ridges on the inner surface of the mantle. Below them, on the body, are the cartilaginous grooves into which the ridges lock when the mantle contracts. The mantle is composed of mainly circular muscle and when they contract the mantles edge is sealed against the body and any slippage is prevented by these lock and key structures. The result is all the water passes out through the funnel. The third projection on the collar is the tip of the chitinous pen, all that remains of the ancestral mollusc shell. It runs to the tip of the posterior surface and protects the soft tissues that lie inside and underneath it. Internal anatomy To expose the visceral mass inside the conical mantle surrounding it, you’ll have to make a longitudinal cut up and through the mantle on the side with the funnel. If you’re using commercially available injected specimens there will already be a cut in the mantle where the various pigments have been injected to enhance the appearance of the circulatory system, just extend that opening. It’s good practice in dissection to never cut along the exact midline so as you make yours do it just off to the side. Why do you never cut along the exact midline of a specimen? Fig. 15. Major internal features of a squid. © BIODIDAC Mantle cavity Like all soft bodied animals it’s going to be easier to see the various systems if they are supported by water. Place you specimen in a dissection tray, pin the sides of the mantle back and flood the specimen with water. By opening the mantle cavity on this side you’ll see that the visceral mass seems to be floating inside the mantle cavity. Move it to either side and you’ll see it’s attached to the mantle by a fine ligament on the underside of the body. It’s on that side where you’ll find the pen, the remnants of the shell. On the inside of the mantle Phylum Mollusca - 14 - Digital Zoology Lab Manual © Houseman surface the pen forms a protective concave structure surrounding the soft body parts underneath it. In our preliminary observations we’ll identify some of the main structures inside the mantle cavity before looking at individual systems in detail. Fig. 16. Lateral view of the head and mantle in the squid. © BIODIDAC Split open the funnel and trace the way that water flows through the mantle cavity. Water enters through the open collar. This is sealed against the body when the circular muscles of the mantle contract, and force the water out through the funnel. The large funnel retractor muscles, combined with the circular and longitudinal muscles in the funnel, direct the jet of water from the funnel; controlling the direction the squid swims. Inside the funnel is a muscular valve that prevents water from entering through the funnel. Underneath the funnel retractors are the cephalic retractor muscles. The mantle is primarily circular muscle, but there is a smaller set of longitudinal muscles that enlarge the mantle cavity. Why are there more circular muscles compared to the longitudinal? The visceral mass is covered by a thin, transparent membrane; the body wall. The paired ctenidia are anchored to the wall of the mantle cavity and where they connect with the body you will see the paired branchial hearts. The rectum and anus are located next to the funnel and the opening is shared with the ink sac that lies along side this part of the digestive tract. What is the function of ink? Underneath the rectum, and between the retractor muscles is the large liver, a modified portion of the digestive gland. Reproductive system Identify the sex of your specimen, and be sure to see both. We’ll make some preliminary observations now. Some of the ducts of the system will be easier to see once observations of the circulatory system are complete and the branchial hearts have been removed. If you have a reproductive female enlarged, paired nidamental glands lie on top of the viscera near the center of the body, and the single ovary, filled with granular eggs is located at that posterior tip of the body. The single oviduct runs along the left side of the body and opens into the mantle cavity through the oviducal funnels. In the area where the oviduct passes under the gills it enlarges forming an oviducal gland. Gently remove the nidamental gland to reveal the oviduct and the opening of the oviduct underneath. Removing the nidamental gland will also expose the accessory nidamental glands underneath. The oviducal gland adds the shell to the eggs and the nidamental glands add the gelatinous capsule that covers the egg case. In the male the single testis appears as a tubular structure just off center and near the posterior end of the squid. The testis lies inside a thin membranous sac and the twisted sperm duct drains the capsule. There is no direct connection between the two and sperm pass into the space of the capsule and then into the sperm duct. A larger convoluted, spermatophoric gland may be visible now, or after the branchial hearts have been removed. The spermatophoric gland Phylum Mollusca - 15 - Digital Zoology LabManual © Houseman packages the sperm into the spermatophore which is then passed to the female by the hectoctylus arm. At the anterior end, locate the penis which lies to the left and behind the rectum. Excretory system. If the delicate body wall remains intact, locate the two openings to the metanephridia. The triangular metanephridia are in front of the branchial hearts. The metanephridia are next to the anterior vena cava, and if you have an injected specimen, the injected dye may make the excretory structures hard to see. The metanephridia filter the fluid in the pericardial cavity. Circulatory and respiratory system Unlike other molluscs, the cephalopod circulatory system is closed. We’ve already located the paired branchial hearts, so let’s start our observations of the circulatory system there. The branchial hearts collect blood from the body through the single, large anterior vena cava that splits into left and right precava that pass by the kidney before entering the branchial hearts. The paired posterior vena cava also drain into the branchial hearts along with left and right mantle veins. Each branchial heart pumps blood into the ctenedia on the corresponding side of the body. Blood enters the afferent branchial artery, crosses the gills where it is oxygenated, and leaves through the efferent branchial veins that connect with the single systemic heart. Fig. 17. Major blood vessels in the region of the branchial and system hearts of the squid. © BIODIDAC Open the pericardial cavity surrounding the systemic heart that pumps oxygenated blood from the ctenidia through the anterior aorta to the front of the body. The posterior aorta supplies the posterior part of the body and divides into three mantle arteries that disappear into the wall of the mantle. Smaller branches of the anterior aorta may be visible supplying the rectum and reproductive structures. Digestive system The digestive system is complex and it’s worth remembering the general mollusc digestive plan. The simplest description is a long tubular gut with a blind ended sac, the gastric or digestive gland. The same applies with the squid, the difference being the digestive Phylum Mollusca - 16 - Digital Zoology Lab Manual © Houseman gland itself has modified into its own separate compartments, and of course these are all going to have names. Morphologists have often used common vertebrate terms to describe these divisions; pancreas and liver are only modified regions of the ancestral digestive gland. Fig. 18. Major features of the digestive tract of a squid. © BIODIDAC Carefully remove the heart and metanephridia without destroying the underlying digestive organs. Underneath the heart is the U shaped pancreas with a granular appearance. The pancreas is part of the duct that leads to the liver. The liver occupies most of the anterior viscera and embedded on its side are salivary glands. Loosen and remove the connective tissue surrounding the liver and gently lift it up to reveal the esophagus and anterior aorta that pass through the region. An equally large structure, this time forming most of the posterior end of the viscera is the caecum, or stomach pouch. This thin walled structure fills most of the posterior part of the mantle cavity and is filled with recently ingested food and secretions of the liver. Other obvious features are the anus and the intestine leading to it. Free the funnel from underlying tissue by cutting the two small siphon protractor muscles between head and funnel and lift the siphon out of the way. Cut through the head between the two arms immediately underneath the funnel to expose the pharynx modified into the buccal bulb. The large, tough, interlocking jaws are easy to identify. Pry them apart and inside you will find that the ancestral radula is still present. Under the radua is the ligula and the two structures are referred to as the odontophore. The esophagus leads out of the bulb, extends through the liver, and connects with the stomach. The stomach has a muscular, thick wall and connects to the caecum, or stomach pouch and its own diverticulum. The pouch in turn is connected to the liver through the pancreas. The intestine is connected to the stomach near the entrance of the esophagus. Nervous system The generalized mollusc body plan has a variety of ganglia positioned in different parts of the body. In the squid these have all become fused and encased in a cartilaginous brain case, or skull. The only exception to this are the stellate ganglia on the inside of the mantle at the tips of the gills. To see the brain cut lengthwise through the head. It will be hard to distinguish the differences but the supraesophagial ganglion is a single paired ganglia and the mass of nerve tissue underneath the esophagus, the subesophagial ganglia, is a fusion of the pedal and visceral ganglia. Circumesophagial connectives connect ganglia above and below the esophagus. The large eyes are connected to the central nervous system through the optic nerves attached to the ganglia underneath the esophagus. Phylum Mollusca - 17 - Digital Zoology LabManual © Houseman