Laboratory Laboratory
10
Vertebrata
152 10: Vertebrates
OBJECTIVES
After completing this lab you should be able to: 1. Compare this vertebrate with the previously studied Phylla in this lab with regards to: body symmetry body cavity support circulation gas exchange feeding and digestion reproduction and development
LAB PREPARATION
In preparating for this laboratory you should do the following: 1. Review this lab. (If you are confused by the anatomical or physiological descriptions in this manual, please refer to the applicable sections in Chapters 41-49 in Campbell, 7th Ed.) 2. Read Chapter 34 in Campbell, 7 th Ed 3. Bring personal protective gear (lab coat, goggles, gloves) to lab.
INTRODUCTION
Frogs are members of the Class Amphibia, Subphylum Vertebrata, in the Phylum Chordata (L. chorda, cord). There are seven extant classes in the subphylum Vertebrata: Agnatha (lamprey and hagfish), Chondrichthyes (sharks, rays, and chimera), Osteichthyes (bony fishes), Amphibia (frogs, toads, salamanders, and caecilians), Reptilia (snakes, lizards, crocodiles, alligators, and tuatara), Aves (birds), and Mammalia (mammals). A. Support & Movement One of the major advantages vertebrates have over other animals is the possession of an internal skeleton (endoskeleton).Invertebrate exoskeletons, because of architectural restrictions, place a limit on the ultimate size of the animal. Exoskeletons also, in some cases, are so heavy as to restrict movement. Vertebrates, on the other hand, with their endoskeleton have nearly unrestricted development in size, e.g., whales and elephants. Furthermore, the relative lightness of the vertebrate skeleton coupled with its strength enable even the largest vertebrates great flexibility and mobility. Vertebrate skeletons are composed of two important materials, bone and cartilage. In fact,the majority of the skeleton of some fish such as sharks is made up of cartilage. Vertebrate bone is formed in one of two ways. It can either be formed directly by mesenchyme cells or it can form secondarily to replace a
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Figure 10-1. Views of the frog skull, from top to bottom: dorsal, ventral, and lateral.
154 Laboratory 10: Vertebrates cartilage precursor. The former type of bone is called dermal or membranous bone and is of mesodermal origin. The latter type is termed endochondral bone and may be of either mesodermal or ectodermal origin. Dermal bone is commonly found in fishes, particularly ancestral types. There it makes up a major portion of the head skeleton, as well as being the principle constituent of the primitive placoid, cosmoid, and ganoid scale types. In terrestrial classes of vertebrates the dermal skeleton is reduced. Those elements remaining in the frog are restricted to portions of the skull, jaws, and pectoral girdle. Endochondral bone of ectodermal origin is collectively called the visceral skeleton. It arises from a special part of the ectoderm referred to as the neural crest. Primitively and embryonically, the visceral skeleton was a support structure for the gills. Since frogs do not have gills the visceral skeleton has been greatly reduced and modified. Remaining elements include the hyoid apparatus, Meckels cartilage, the laryngeal skeleton, tracheal cartilages, and a small bone in the ear called the columella. The majority of the frog skeleton is made up of endochondral bone of mesodermal origin. This includes much of the skull, the vertebral column, the pectoral and pelvic girdles and limbs, and the sternum. The vertebrate skeleton can be divided a number of ways based on form, structure, function, position, or embryonic origin. While no method is absolutely satisfactory, the following scheme will be used here for the frog. B. Bullfrog Axial Skeleton 1. The Skull (Refer to Figures 10-1a, b, & c.) The skull includes the chondrocranium, or braincase, made up of many individual bones of dermal and endochondral origin, and the jaws. The chondrocranium has numerous openings for the passage of nerves and blood vessels, e.g., the foramen magnum for the spinal cord, and the placement of sense organs, e.g., the orbits for the eyes. It also encloses and supports the ear, or auditory capsule. The upper jaw consists of the premaxillary, maxillary, and quadratojugal bones. The lower jaw is made up of the small Meckels cartilage surrounded by the dentary and angulare bones.
The Hyoid Apparatus The hyoid apparatus (absent in our prepared skeletons) is located in the floor of the oral cavity. It is cartilaginous and supports the tongue and larynx.
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Figure 10-2. Frog vertebral column and the urostyle: close-up of a vertebra. The Vertebral Column (Figure 10-2) The vertebral column of the frog consists of nine vertebrae and the urostyle. The first vertebrae, the atlas, articulates with the occipital condyles of the skull. The ninth vertebrae, called the sacral vertebrae, has transverse processes which articulate with the ilia of the pelvic girdle. Most vertebrae, regardless of their location, fit a general plan. Each consists of a body or centrum which articulates with the other vertebrae, a neural spine for the attachment of muscles, transverse processes for the attachment of muscles and articulation with the ribs, articular processes which articulate with the other vertebrae, and a vertebral foramen for the passage of the spinal cord. Sternum The sternum protects the heart, lungs, and other internal organs as well as providing a site for muscle attachment. In the frog it is composed of five parts: the episternum, omosternum, epicoracoid, mesosternum, and xiphisternum. Ribs The frog does not have ribs. Based on embryological studies, some vertebrate biologists believe that frogs do have small ribs that fuse with the transverse processes of the vertebral column.
156 Laboratory 10: Vertebrates C. Bullfrog Appendicular Skeleton Most vertebrates possess some type of paired appendages be they fins, arms, wings, or flippers. Whatever the modification, all are built from the same basic plan of a girdle articulating with the axial skeleton and limbs articulating with the girdle. Among terrestrial vertebrate classes there are even greater similarities, with many of the appendicular bones being homologous (derived from a common embryological precursor) and possessing the same name . Pectoral Girdle & Limbs (Figure 10-3a.) Each half of the pectoral girdle of the frog includes a suprascapula, scapula (shoulder blade), clavicle (collar bone), and coracoid. Connected to the girdle by means of ligaments at the glenoid fossa is the humerus. The humerus, in turn, articulates with the radioulna (a fusion of the radius and ulna). Out from the radioulna are the carpels, metacarpals, and phalanges. A small medial fifth digit, called the prepollux, also articulates with the carpals. (The medial fifth digit is used by the male frog to hold onto the female frog during mating.) Pelvic Girdle & Limbs (Figure 10-3b.) Each half of the frog pelvic girdle is made up of the ilium, pubis, and ischium. The two halves of the pubis and ischium are fused so that the four bones appear as one. At the junction of the three pelvic girdle bones is a socket, the acetabulum, where the femur attaches. Out from the femur is the tibiofibula (the fusion of the tibia and fibula), the two tarsal bones, astragalus and calcaneum, the metatarsals, and the phalanges. Like the forelimbs, the hindlimbs possess a small medial digit, the prehallux. Its believed that the principle function of this medial digit is to assist in digging.
Laboratory 10: Vertebrates D. Muscles & Muscle Terminology Vertebrate muscles can be categorized as smooth, cardiac, or skeletal based on location, fine structure, and innervation. Your text has a good discussion on the differences among these types and on how muscles contract. This lab will focus on the skeletal muscles which are responsible for locomotion. Skeletal muscle fibers are bound together in bundles called fascicles. Fascicles are, in turn, bound together to form muscles and covered by a connective tissue called fascia. Most muscles are attached either to another muscle or to bone. The attachment is fleshy if the muscle fibers are attached directly to bone. The attachment can also be by means of a cord-like piece of connective tissue called a tendon or by a broad sheet-like piece of connective tissue called an aponeurosis. When a muscle contracts, one end of the muscle remains relatively motionless or fixed while the other end moves, pulling a bone or other structure nearer the fixed point. The fixed end is called the origin of the muscle while the moving end is termed the insertion. The muscle segment inbetween is called the belly. Muscles are commonly arranged in opposing pairs or groups. Below is a list of the common types of opposing muscles. 1. Flexors cause a bending or decrease in the angle between two parts of the body. 2. Extensors cause a straightening or extension of a part of the body. 3. Adductors move a body part towards the midline of the body. 4. Abductors move a body part away from the midline of the body. 5. Depressors lower a body part. 6. Levators raise or elevate a body part. 7. Protractors draw parts forward or out. 8. Retractors retract parts back. 9. Constrictors (or Sphincter) surround an orifice and close it. 10. Dilators surround an orifice and open it. 11. Rotators twist a limb or body part. E. Respiration & Circulation In frogs, the exchange of oxygen and carbon dioxide between blood and air occurs at three different sites: the skin (cutaneous respiration), the lungs (pulmonary respiration), and the mucous membrane of the mouth and pharynx (buccopharyngeal respiration). In each of these sites, the blood in capillaries is separated from the air by only a thin membrane. The relative importance of the three types of respiration varies, depending upon the frogs environment, i.e., in or out of the water, and activity, i.e., resting, hopping, feeding.
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Figure 10-4. Pulmonary respiration in the frog. In frogs, pulmonary and cutaneous respiration have been shown to account for the majority of the oxygen-carbon dioxide exchange. The exchange is small in buccopharyngeal respiration and its believed that the regular oscillatory movements of the mouth floor are primarily to aid olfaction rather than respiration. During buccopharyngeal respiration the lungs remain full of air with the glottis closed. The floor of the mouth is rapidly and regularly raised and lowered, forcing air in and out of the oral cavity through the open nares (nostrils).
Mammals draw air into their lungs with a diaphragm and birds and reptiles do the same with expandable ribs, frog and toads possess neither. How then do frogs and toads breath? Breathing (pulmonary respiration) can be divided into four steps (Figure 10-4). 1. Air is drawn into the oral cavity through the open nares by closing the glottis and depressing the floor of the mouth. 2. The nares are closed, the glottis opened, and the floor of the mouth is raised to force air from the oral cavity into the lungs. 3. Air is expelled from the lungs back into the oral cavity by a) the contraction of body wall muscles, b) elastic recoil of the stretched lungs, and c) depression of the floor of the mouth. 4. The spent air is expelled from the frog by opening the nares, closing the glottis, and raising the floor of the mouth. As air is forced into the lungs (step 2 above) it passes through a short tube, the larynx. The larynx is reinforced by cartilage and contains two elastic bands, the vocal cords. These cords can be vibrated by vigorously forcing air across them from the lungs (step 3) to produce the familiar croaking sounds. Posterior to the larynx are the paired bronchi, short tubes that lead into the lungs. Within the lungs is a network of partitions that divide the lung into many small chambers called alveoli. The alveoli, with their numerous blood capillaries, are the site of gas exchange. In vertebrates, respiratory gases are transported throughout the body via a closed circulatory systems.
Laboratory 10: Vertebrates F. Feeding & Digestion All vertebrates, with the exception of the Class Agnatha, possess jaws. The vertebrate jaw is believed to have evolved from the primitive gill arches of ancient fish. The development of jaws was an important step in the evolution of vertebrates. With jaws, vertebrates were able to expand their means of food gathering. While the early chordates were restricted to filter and deposit feeding, jawed vertebrates could actively pursue, capture, and manipulate their prey. Frogs possess a relatively uncomplicated vertebrate mouth, with an upper jaw (composed of the maxilla, premaxilla and quadratojugal bones), a lower jaw, the mandible (made up of Meckels cartilage, and the dentary and angulare bones), and simple dentition (maxillary and vomerine teeth). A frog catches most of its food (a variety of living prey including insects, worms, spiders, and smaller amphibians and reptiles) with its tongue. The tongue is attached to the anterior floor of the oral cavity. Its posterior end is sticky due to secretions from glands located in the roof of the oral cavity. To catch its prey, the frog rapidly flips its tongue out of its extends mouth, it, and curls it around the animal. Stuck to the tongue, the animal is quickly pulled back into the frogs mouth. To swallow its food the frog closes its glottis, raises the floor of its mouth, and contracts its eyes down into the oral cavity. The result is a reduction in the volume of the oral cavity, forcing the food into the esophagus. The frog will often wipe its mouth with its forelimbs to force any loose appendages into the mouth. From the esophagus, food passes into the stomach where digestion begins. Gastric juices break down proteins and fats, while circular muscles of the stomach contract to break the food into smaller pieces. A pyloric valve controls the passage of food from the stomach to the small intestine. In the small intestine, the food is further digested and absorbed. The intestine is divided into a short duodenum and a much longer, coiled ileum. In addition to its own digestive secretions, the small intestine receives digestive fluids from two large digestive organs, the liver and the pancreas. The liver's primary digestive function is the production of bile, an alkaline fat emulsifier. Bile is stored in the gallbladder and is periodically released it into the bile duct. The pancreas secretes its digestive fluids into a pancreatic duct which later joins the bile duct, now called the common bile duct. The common bile duct empties into the duodenum.The pancreatic fluids contain digestive enzymes involved in the breakdown of fats, carbohydrates, proteins, and nucleic acids.
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160 Laboratory 10: Vertebrates The small intestine leads into the large intestine or colon. The primary function of the colon is the absorption of water and ions from the colon lumen into the blood stream. It is also a temporary storage area for undigested fecal matter. The colon passes posteriorly through the pelvis into the cloaca. The cloaca is the terminal part of the frogs digestive tract receiving the waste products of the digestive and urinary systems as well as the reproductive products (eggs and sperm). G. Reproduction and Development Frogs are gonochoric, but some may change sex at a certain age. Fertilization is external and females usually lay numerous eggs which are attached to the bottom of streams or ponds. Like all vertebrates, frogs are deuterostomes. Development may be indirect, with a distinct larval (tadpole) phase in most species.
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LABORATORY EXPERIMENTS
Students should work in pairs for all exercises. A. External Anatomy 1. Each pair will be provided with a preserved frog. 2. Identify the external features highlighted in Figure 10-5. Note that the toad has only a middle and inner ear and lacks the external ears common in mammals.
Figure 10-5. External anatomy of the toad. B. Dissection of grass frog
Students must use personal protective gear (lab coat, goggles, gloves) when handling preserved specimens . 1. Place your frog in your dissection tray, ventral surface up. 2. 3. Open the mouth with your blunt probe in order to identify the features highlighted in Figure 10-6. Cut through the angle of the jaw with your scissors (about 1 cm on each side), as shown in the figure, to better observe the interior of the mouth. 4. Locate the maxillary teeth. With your probe explore the various openings including the glottis and vocal sacs. The eustachian tubes are passages to the middle ear.
162 Laboratory 10: Vertebrates 5. Posterior to the attachment of the tongue you should feel a hardened plate-like structure on the floor of the mouth not depicted in Figure 10-6; this is the hyoid apparatus. With your probe investigate its size and shape.
Figure 10-6. The oral cavity of the frog. Turn your attention now to the internal organs of the frog. To investigate these it will be necessary to open up the abdominal cavity. 1. First, however, you should remove the skin of your frog to expose the abdominal muscles (Figure 10-7). Do this by first pinching up a fold of skin in the center of the frogs ventral abdominal wall. Using your small scissors, cut through the fold to make a small incision. 2. Extend this incision forward to the back of the mouth and back to the junction of the hindlimbs. Now make two transverse incisions, one just posterior of the forelimbs, and the other anterior to the hindlimbs. 3. Fold back the skin to expose the underlying muscles. Use your small scissors and forceps to sever the blood vessels and fascia connecting the skin to the muscles. Several skeletal features should now be visible: the sternum, clavicle and coracoid bones. 4. Cut through the ventral abdominal musculature by duplicating the procedure you used for removing the skin.
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Figure 10-7. The procedure used to dissect into the abdominal cavity. Be careful not to cut too deeply or you will damage the underlying internal organs.
5. To open up the thoracic region it will be necessary for you to cut through the coracoid and clavicle. Do this with your large scissors. If done successfully, your dissected frog should look similar to Figure 10-8. 6. Begin examining and identifying the organs and structures highlighted in Figure 10-8. 7. One of the most conspicuous organs inside the frog is the large reddish-brown liver. What digestive function does this organ serve? Notice that it is made up of three separate lobes. Lift it up and you should discover a small greenish sac, the gallbladder, attached to its dorsal surface near the centrally located heart. 8. Also rather obvious is the stomach located on the left side of the animal. Follow it down and identify the pyloric valve, duodenum, ileum and colon. You will notice that the stomach and intestine are supported by a thin transparent sheet, the mesentery. The mesentery is derived from the peritoneum which lines the entire coelom and gut. Within the mesentery, between the duodenum and stomach you should locate the diffuse reddish or
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Figure 10-8. Ventral view of the frog's digestive tract and internal organs.
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Figure 10-8. Ventral view of the frog's digestive tract and internal organs (actual dissection).
166 Laboratory 10: Vertebrates whitish organ, the pancreas. What is the digestive function of this organ? Because the lungs deflate after death, their relative size is deceptive. Search for them dorsal to the liver. Note the network of blood vessels crisscrossing them. 9. Follow the lungs forward. While the bronchi are obscured by the blood vessels of the heart and difficult to see, the larynx is not. It is the hard whitish structure just anterior to the heart. Cut through the several small muscles covering and controlling the larynx to better reveal its appearance. In addition to the above structures there are some others, not directly associated with digestion or respiration, which are easily identifiable. These include; the small red spleen anterior to the stomach, the bright yellow fat bodies, the transparent urinary bladder, and the dorsally located kidneys and testes (male) or ovaries (female). (You may have noticed the lack of subcutaneous fat when you removed the frogs skin; thats because there is none. Unlike humans, frogs can only get big - not fat.) By pushing all the internal organs to one side you can see the vertebral column, urostyle, and ilia. Also visible are some of the vertebral nerves that innervate the hind legs. You can identify a vertebrate nerve by its shiny white appearance and tough stringy texture; both are a result of the myelin sheath which enwraps the nerve. C. Circulatory system: ventral view 1. In the rear of the lab is a large specimen in which arteries (carrying blood from the heart) have been injected with red latex and veins (carrying blood toward the heart) have been injected with blue latex. 2. Examine this demonstration dissection and identify the components of the circulatory system highlighted in the text below. Draw a sketch of this dissection, with the highlighted components labeled. 3. The heart of the frog and other amphibians has three chambers two atria (singular, atrium) and one ventricle. The heart is enclosed within the pericardial cavity, which has been opened up in this dissection to better reveal the heart. 4. Leading from the ventricle is the conus arteriosus, which splits into left and right branches of the truncus arteriosus.
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Figure 10- . Closeup of the heart and related arterial system. White is the ventricle while yellow and green show the auricles. 5. The ventral abdominal vein is one of the important veins that collects blood from the abdominal organs and returns it to the heart. The ventral abdominal vein carries blood from the pelvic region to the liver. There it divides into another capillary bed before continuing to the heart. 6. Note, also, the highly vascularized skin. When the frog is submerged in water, gas exchange takes place through the skin and the lining of the oral cavity. At low temperatures there is abundant dissolved oxygen in the water and frogs can remain submerged for long periods of time. In warm water, however, the dissolved oxygen content is much lower and frogs must periodically come to the surface to supplement their supply of oxygen by gulping air into their lungs. D. Skeleton 1. Take a break from your frog and examine one of the prepared skeletons in the lab. The skeleton is that of a bullfrog and all the bones are the same as those in your frog, the only difference being one of relative size. Be sure you are able to distinguish the axial skeleton from the appendicular skeleton. Also take a look at the individual vertebrae present in the laboratory and identify the centrum, transverse and articular
168 Laboratory 10: Vertebrates processes, neural spine, and vertebral foramen. What is the function of each of these vertebral parts? E. Musculature The final part of todays exercise is the examination of the frogs hindlimb musculature. 1. Remove the skin from the right leg of your frog with your small scissors and forceps by making an incision along the leg and out toward the foot. 2. Expand the incision, being careful not to damage the muscles underneath. In some areas, such as near the femur-tibiofibula joint (the knee), the fascia will be especially prevalent; be particularly careful in these areas. 3. Remove the skin from the entire leg (except for the end of the foot) and place the frog dorsal surface up. 4. Begin your examination of the dorsal muscles, refering to Table 10-1 and Figure 10-9. You will notice that all the muscles are enclosed in a tough transparent sheath of connective tissue. Before penetrating this sheath quickly examine the muscles that are visible through it and make some tentative identifications. To confirm these identi-fications you must remove the sheath. Do this with your small scissors and forceps, cutting along the lines you believe separate the muscles you have identified. It is very important that you be careful with your separations. Each muscle is itself enclosed in a connective tissue sheath; if you can not see this sheath you are probably cutting through the muscle rather than separating it. 5. By referring to Table 10-1 identify the origin(s) and insertion(s) of the triceps femoris, biceps femoris, semimembranosus, gracilis minor, gastrocnemius, and peroneus. If necessary, refer to the demonstration dissection of a large specimen in the back of the room to confirm your identifications.
6. When you have finished with the dorsal muscles, place your specimen ventral side up. 7. Referring to Figure 10-10 and Table 10-2, identify the gracilis major, adductor magnus, and sartorius. Periodically refer back to the prepared skeleton to help understand the function of each muscle. If you have time, remove the skin from the frogs left leg and test your ability to identify the capitalized muscles in Table 10-1.
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LAB SUMMARY
1. Complete Table 10-3, keeping your entries as short as possible. (Note that the table covers 2 pages, and will be a useful review for your final lab exam.) 2. Answer the following discussion questions in 1-2 sentences: a.What are the advantages and disadvantages of an endoskeleton, relative to an exoskeleton? b.What are the major components of the frogs axial skeleton? Appendicular skeleton? c.List three pairs of opposing muscles and their functions. d.What are the three types of respiration utilized by the frog, and at what sites does gas exchange take place for each? e.Briefly describe the frogs digestive system and associated organs. f.Compare and contrast muscle action and attachment sites in insects (exoskeleton) and frogs (endoskeleton).
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Platyhelminthes (flatworms) Body Symmetry
Annelida (earthworms)
Mollusca (clams)
Body cavity (coelom)
Support (skeleton)
Circulation
Gas Exchange
Feeding & Digestion
Reproduction & Development
Laboratory 10: Vertebrates
Arthropoda (cockroaches) Body Symmetry Echinodermata (starfish)
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Chordata (frogs)
Body cavity (coelom)
Support (skeleton)
Circulation
Gas Exchange
Feeding & Digestion
Reproduction & Development
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