Biology 4B Laboratory Invertebrates II Page 1 of 7

Biology 4B Laboratory Invertebrates II: Mollusca, Annelida and Nematoda Objectives

To understand the basic differences among the invertebrate animal phyla

To investigate and learn the obvious external and internal characteristics of annelids, nematodes, arthropods and echinoderms

To investigate at the microscopic level the organization and function of selected tissues and cells within these groups

Figure 1. Cladogram of the Major Animal Phyla based upon SSU-rRNA

INTRODUCTION In this laboratory, we will survey three of remaining five invertebrate phyla: Mollusca, Annelida and Nematoda (Figure 1). We have only studied one of the five major protostome phyla. Of the remaining major phyla of protostomes in which we will study in this laboratory, only the annelids exhibit metamerism, the division of the body into segments (you will study arthropods in the Inverts III laboratory). Segmentation is advantageous during development, where greater efficiency is obtained by constructing a whole organism out of identical somites or segments. In the adult, locomotor activity is enhanced because of the independent nature of each segment and the flexibility afforded by a series of segmented parts. Segmentation also gives these phyla a survival advantage. Since many segments are similar to other segments in form and function, damage to one or several segments does not necessarily compromise body functions. PHYLUM MOLLUSCA Triploblastic eucoelomate animals The molluscs are classified as triploblastic eucoelomate animals, as are all phyla remaining to be examined. Most molluscs are bilaterally symmetrical and have well-defined circulatory, respiratory, excretory, and digestive systems. With nearly 50,000 species, the molluscs are a large group, second only to arthropods. The name "mollusc" is derived

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from the Latin molluscus ("soft"), indicating that the molluscs are soft bodied animals. The group includes the snails, bivalves, chitons, squid, octopuses, and others. In some forms, the soft bodies are protected by a calcareous shell. The organisms in the phylum Mollusca are characterized by having three main body areas: a head-foot (sensory and

locomotion structures), a visceral mass (excretory, digestive, and circulatory structures), and a mantle (which secretes the shell). The gills, which function in respiration, are located between the visceral mass and the mantle. In this lab we will look at four classes: Class Polyplacophora, the chitons; Class Gastropoda, the snails and slugs; Class Bivalvia, the clams, oysters, and allies; Class Cephalopoda, the squid, octopus, and chambered nautilus. Class Polyplacophora This class is considered the least advanced of the phylum. These animals are entirely marine, and have oval bodies with a shell consisting of eight dorsal plates. A broad, flat foot used in locomotion is located ventrally. The mantle cavity is reduced to a groove running on either side of the body between the foot and the margin of the animal. Examine the chitons on display. In some cases you will see only the

eight dorsal plates diagnostic of the class. In the preserved jars you will also observe the ventral foot and perhaps the mouth on the anterior end (Figure 12.)

Class Gastropoda This class includes snails and slugs. It is by far the largest class of molluscs. Gastropods are primarily marine, but some species also inhabit freshwater and terrestrial habitats. In all forms, the visceral mass is located enclosed in a coiled shell during early developmental stages. In most gastropod species the shell is retained in the adult, but in some, such as the common garden slug, it has been completely lost. Because of this, slugs are restricted to moist areas to prevent desiccation. Observe several of the snail shells in present in the lab. Notice the type of coiling and external decoration of these

shells. Observe the slug, nudibranchs and other gastropod specimens in the lab Class Bivalvia Members of this class are characterized by a shell consisting of two valves or halves. Bivalves use a muscular foot for locomotion. Siphons are used to draw in a stream of water which is passed over the gills for feeding and respiratory purposes. You will now complete a as directed below. Examine the clam externally. Find the two valves, the hinge ligament that holds them together, the swollen umbo at

the anterior end of the hinge, and the lines of growth.

Using the position of the hinge and umbo, determine which side is dorsal, which side is ventral which end is anterior, and which end is posterior.

Internal Clam Structures (Figure 14): Locate the position of the anterior

adductor muscle and posterior adductor muscle through the narrow opening between the valves. Slip a scalpel between the mantle and the left valve. Use the scalpel to gently pry the adductor muscles away from the valve to which they are attached. Loosen the mantle

Figure 12. Chiton foot anatomy

Figure 13. Internal shell anatomy of clam

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over the entire area of the left valve and open the clam. Examine the inner surface of the empty valve. Notice its smooth nacreous surface. Observe the position of the

various muscle scars, including anterior adductor muscle scar, posterior adductor muscle scar, anterior protractor muscle scar, anterior foot retractor muscle scar, and posterior foot retractor muscle scar. Also note the pallial line, which is the point where the mantle attaches to the shell (Figure 13).

Look at the mantle, including the pallial muscle. The mantle of the left and right valves comes together posteriorly to form a ventral incurrent aperture and a dorsal excurrent aperture that allow water to enter and exit the mantle cavity. Water enters through the incurrent aperture and leaves through the excurrent aperture.

The space between the mantle and the body is the mantle cavity. Lift the mantle to expose the visceral mass, foot,

gills, and associated structures. The muscular, wedge-shaped foot is at the ventral aspect of the body. The soft tissue making up the bulk of the body is the visceral mass. Between the mantle and the visceral mass lie two gills. At the anterior margin of the visceral mass, note the smaller, flap-like, labial palps. Labial paIps surround, and direct food toward, the mouth. Water coming in from the incurrent aperture reaches the ventral aspect of the gills and passes dorsally through the gills into a suprabranchial chamber. Water is then directed posteriorly and out of the mantle cavity through the excurrent aperture. In the process, suspended food particles are filtered and gas exchange occurs. Food particles are transported by cilia to food grooves along the dorsal margin of the gills. Cilia in the food grooves transport food to the labial palps.

Return the mantle to its original position and locate the pericardium, a thin membrane dorsal to the visceral mass. The circulatory system of bivalves is an open system in which blood leaving the heart flows freely between the organs. Carefully open the pericardium to see the heart. The heart wraps around the intestine where the intestine emerges from the visceral mass. The intestine is running posteriorly to empty at the excurrent aperture. The heart consists of two parts, a thick-walled ventricle surrounding the intestine and two thin-walled auricles attached at either side of the ventricle. If you were careful in removing the pericardium, you should see both. Look for a dark mass of tissue ventral to the pericardial sac. This is an excretory organ known as a nephridium. Nephridia remove metabolic waste products from the blood and release the waste into the mantle cavity near the excurrent aperture. Finally, use your scalpel to make a sagittal section of the foot and visceral mass. Within the visceral mass note the cut sections of intestine. The yellowish-brown tissue surrounding the intestine is gonad.

Class Cephalopoda The cephalopods are considered to be the most advanced class of molluscs. These organisms have a highly evolved visual system, and tentacles with suction cups. They are all marine. They are fast swimmers and use jet propulsion as a means of locomotion (Figure 15).

Figure 14. Internal anatomy of the clam

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Examine the preserved squid (Loligo). Find the following structures: eight arms, two tentacles, mantle (enclosing the

visceral mass), lateral fins, eyes, located just anterior to the mantle, siphon - protruding from below the mantle. Water drawn into the mantle cavity can be forcefully expelled through the siphon when muscles of the mantle contract, resulting in jet propulsion. The siphon can direct the jet of water in different directions.

Compare the external anatomy of an octopus to the squid, noting the difference in the shape of the mantle and the octopus's lack of fins.

PHYLUM ANNELIDA Members in the phylum Annelida are often referred to as segmented worms because of their segmentation, a distinguishing characteristic that sets them apart from other animals. The most recognizable members include the earthworms (terrestrial habitat), leeches (terrestrial and freshwater), and marine worms. All annelids are triploblastic, bilaterally symmetrical, and eucoelomate. In addition, annelids exhibit a body wall with both longitudinal and circular muscle layers (which, along with segmentation mentioned above, allows these animals to be quite mobile). They have a complete digestive tract. Their nervous system shows some degree of cephalization with a “brain” and two ventral nerve cords that running the entire length of the body. They have a closed circulatory system with aortic arches that act

Figure 15. External and Internal anatomy of squid, Loligo

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Figure 16: Structure of a clamworm (Nerius)

Figure 17. Anterior end of the earthworm

as the “heart” to pump blood through muscular blood vessels. They also have a well-developed excretory system which removes waste from the blood and coelom. There are three major classes within the phylum Annelida, described below.

Class Polychaeta - mostly marine worms, such as Nereis (the clamworm) Class Hirudinea - the leeches (predominantly freshwater), such as Hirudo Class Oligochaeta - mostly freshwater and terrestrial worms, such as Lumbricus (the earthworms)

CLASS POLYCHAETA Polychaete worms are mostly a marine group of worms characterized by many segments with a pair of parapodia with numerous setae (Figure 16). They have a distinct head with eyes, palps and tentacles. Examine a clamworm (Nerius). These are the

“typical” polychaete worms that can be found living in the mud and debris of shallow coastal waters. Using the dissecting scope, observe the head region and find the following: eyes, mouth on the ventral side, jaws, and tentacles.

Examine one of the segments. Locate a parapodium on one side a body segment. Parapodia function in locomotion and respiration for polychaetes. Each parapodium is comprised of two lobes which bear numerous setae (the reason for the class name).

CLASS OLIGOCHAETA Like polychaete worms, oligochaete worms are also segmented both outside and inside. However, oligochaetes do not have parapodia, their head is less developed and they have fewer setae. The most noticeable external feature in this group is the clitellum (figure 3). Most members in this class are either terrestrial (most) or inhabit freshwater. Obtain an earthworm (Lumbricus) and place the animal in a dissecting tray. You may need a dissecting scope to fully

appreciate the external anatomy. The first four segments comprise the head region. Find the mouth on the first segment (Figure 17). The prostomium overhangs the mouth. They have a complete digestive tract that terminates on the last segment with the anus.

The most obvious external feature is the clitellum, a swollen area in

the anterior third of the specimen. This region functions in reproduction by secreting a mucous which holds the participants together during sperm exchange and cocoon formation around the fertilized eggs.

Orient the worm dorso-ventrally by locating the tiny setae (hairs).

Run your fingers along the animal to feel the rough texture produced by the setae. Four of these structures are found on the ventral surface of each metamere. They provide traction during locomotion.

CLASS HIRUDINEA The best known member in this class is the freshwater leech. Other members can be found on land and the marine environments. Members in this class typically have 33-34 segments with a clitellum. Most do not have setae and no members have parapodia. Members have both anterior and posterior suckers.

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Figure 4: Comparison of body cavities.

Examine representative members in this class. The medicinal leech, Hirudo medicinalis, secretes an anticoagulant on the host they parasitized them. This leech was commonly used in the practice of blood-letting. It is still used today to increase circulation to surgical areas, especially with finger reattachments. Note the smaller oral sucker and larger posterior sucker.

PHYLUM NEMATODA Triploblastic pseudocoelomates The development of a body cavity (coelom) is considered a major evolutionary advantage over those animals which do not possess a body cavity (acoelomate). As you have already learned, body cavities are advantageous for a number of reasons, such as to provide more room for organ development, to provide an increased surface area for diffusion of gases and/or nutrients, and to facilitate locomotion by serving as hydrostatic skeleton. A body cavity (Figure 18) is characteristic of all bilateral animals above the acoelomates. A true coelom is a cavity in which the inner body wall and the visceral organs are lined with peritoneum. A pseudocoeIom, found in animals to be examined in the present exercise, is defined as a body cavity that is lined by mesoderm externally and endoderm internally (figure 4).

The nematodes are one of several phyla usually discussed together as the pseudocoelomates because of their shared possession of this structure. Except for this one common feature, they are a diverse group of animals, only distantly related. Included in this broad group of animals are the Phyla Rotifera, Nematomorpha, Gastrotricha, Kinorhyncha, and others. We will examine members of the phylum Nematoda as a representative pseudocoelomate. However, nematodes are grouped with arthropods as an ectodyzoan due to molecular evidence supporting ecdysis, the ability to shed the exoskeleton as the organism grows. Nematodes have a worldwide distribution that include, terrestrial, freshwater, marine and parasitic forms. They are round worms with a tough, flexible cuticle that is non;living. Nematodes are important ecologically for their recycling and decomposition capabilities. OBSERVATION OF NEMATODA Examine the male and female intestinal roundworm, Ascaris lumbricoides, in dissecting tray. Gloves should be worn, if available. If not, handle organism with forceps. Do not touch with bare skin. Ascaris is sexually dimorphic and sexes are easily differentiated. Male worms are smaller, typically have a hook-shaped sideways bend near their posterior end, and may have tiny copulatory spicules protruding slightly from the cloaca. Ascaris is an intestinal parasite of vertebrates, with A. lumbricoides infecting up to 64% of individuals living Figure 18. Triploblastic body plans: acoelomate,

pseudocoelomate and coelomate.

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in the southeastern United States. The female lays up to 200,000 eggs/day which passes out of the host via their feces. The embryo is very resistant, thus be careful handling these worms as you could potentially infect yourself.

Examine the slide of a trichina worm, Trichinella spiralis. You will be observing calcified cyst of the juvenile trichina

worm (Figure 19) in the muscle of the host. Trichinosis is the disease that is caused by the trichina worm. Human infestations are typically due to the ingestion of undercooked meats such as pork. Roughly 2% of the population in the US has a light infection with trichina worms. Heavy infestations may cause death. Other species that can be infected include: hogs, rats, dogs, cats and any other omnivorous or carnivorous species.

Figure 19: Encysted juvenile Trichinella spiralis

Examine the slide of a hookworm, Necator americanus. Note the anterior portion of the worm with a hook-like

appearance in a tissue section. Infestations results when the juvenile hookworm comes in contact with the skin and burrows into the host. The juvenile then travels via the bloodstream to the lungs, move up the respiratory tract and then swallowed. In the small intestines they will mature.

Examine a slide of pinworms, Enterobius vermicularis. These worms live in the large intestines of humans and are

the most common nematode parasite. These infestations are more embarrassing than debilitating. The female will travel to the anus at night to deposit eggs around the anus. Scratching contaminates the hands and bedding. The eggs are then swallowed and hatch in the duodenum and mature in the large intestines. This is a very common infestation in children.

Examine the free-living nematode, the vinegar eel (Tubatrix aceti) on a depression slide, if available. These worms

have a high tolerance to low pH. Describe how the body bends.