1 Bi 124.1 (Parasitology) Prepared by: Dr. Merab A.

. Chan EXERCISE 9 PHYLUM NEMATODA Introduction A nematode is characterized by an extremely tough thick cuticle externally, and a very high hydrostatic pressure within. The phylum is unusually ubiquitous. The nematodes are free-living in marine, freshwater, and land habitats; and parasitic in animals and plants. Nematodes are slender worms, circular in cross-section and ranging in length from 200 m to 40 cm (in Ascaris, a large intestinal human parasite) or even nine meters in a parasite of whales. The worms are triploblastic, unsegmented and enclosed in a tough but flexible cuticle, under which is a layer of longitudinal muscle. There is no blood or other circulatory system. The body cavity is termed a pseudocoel, since it is probably derived directly from the blastocoel with no inner lining of mesoderm. It contains many tubes of the reproductive system and also the gut, which opens at both ends of the worm. The life cycles of nematodes are of two basic types: direct or monoxenous (i.e., with only one host in the cycle) and indirect or heteroxenous (i.e., with two or more hosts in the cycle). The basic pattern of development is similar whether the life cycle is direct or indirect. Larvae (also known as juveniles) hatching from the eggs progress through a series of stages in their development. Beginning with the first stage, each one is separated by a molting of the cuticle. There are four juvenile stages, i.e., first, second, third, and fourth, followed by the adult. The third stage is infective to the final host. The pattern of growth of the juveniles (J) and occurrences of the successive molts (M) may be expressed as follows: Egg J1 + M1 J2 + M2 J3 + M3 J4 +M4 Adult

Eggs of parasitic nematodes vary in shape, size, thickness of shell, and external markings, as well as the stage of development when laid. Each ovum comprises an egg cell that is enclosed in three primary layers, which consist of: 1) the thin vitelline membrane surrounding the egg cell; 2) the middle thick egg shell; and 3) the outer protein layer, which may be stained yellow with bile. In this exercise, the members of the following orders will be studied. Order Trichurida Family Trichuridae Trichuris trichiura Family Trichinellidae Trichinella spiralis Family Capillariidae Capillaria hepatica C. philippinensis

2 Order Rhabditida Family Strongyloididae Strongyloides stercoralis S. ratti Order Strongylida Family Ancylostomidae Necator americanus Ancylostoma duodenale A. caninum Order Ascaridida Family Ascarididae Ascaris lumbricoides A. suum Toxocara canis Family Anisakidae Anisakis spp. Order Oxyurida Family Oxyuridae Enterobius vermicularis Order Spirurida Family Dracunculidae Dracunculus medinensis Objectives At the end of the activity, the students are expected to: 1. identify and describe some members of the Phylum Nematoda; 2. understand the significance of studying the morphology and anatomy of nematodes; and 3. be familiar with some of the parasitic nematodes of man and other animals. Materials a. Compound light h. Cotton balls microscope i. Big jar with cover b. Dissecting microscope j. Dissecting pan c. Dissecting set k. 0.9 % saline solution d. A pair of gloves l. Petri dishes e. Chloroform or ethyl-ether m. Small beakers f. White rat or wild mouse n. Transfer pipettes (one per group of five) o. 12 inches sewing thread g. Dogs feces p. 100 ml drinking soda q. Prepared slides (w.m.) of the following parasites: Trichuris trichiura (adult male and female), Ancylostoma caninum (adult male and female), and Capillaria (male and female). r. Prepared slides of hookworm rhabditiform larvae, hookworm filariform larvae, and Trichinella spiralis larva. s. Prepared slide of ova (Trichuris, Ascaris, and hookworm.

3 t. Preserved specimens of Ascaris suum (adult) u. Prepared slides of cross-section of male and female A. lumbricoides v. Reference materials on the different parasitic nematodes Methodology 1. Studying Live Nematodes a. Trichinella spiralis Obtain a wild/white rat. The animal must be killed with deep anesthesia using either ethyl ether or chloroform before dissection. Once the animal is killed, place it on its back. Make a midventral and two lateral incisions, free the diaphragm, and fold back the body wall to expose the thoracic and abdominal cavities. Using a transfer pipette, transfer any body fluid to a slide and add a cover glass for examination under a compound microscope for young juveniles of T. spiralis. Record all observations; e.g., number of juveniles seen, other structures seen, etc. Ligate the hepatic portal vein near the intestine and the aortic arch above the heart to collect or concentrate the blood in the vital organs, e.g., the lungs, heart, and liver. Then, cut the blood vessels so that the ligatures will keep the blood within the limits bounded by them and remove the three vital organs listed above as a unit to a dish of saline solution, where they should be opened to remove the blood. Sediment and decant until the solution is clear and examine for the juveniles of T. spiralis. Remove some serosa, transfer to a slide, smooth out in a drop of saline solution, add a cover glass, and examine under a compound microscope. Count the number of juveniles seen. Cut several pieces of skeletal muscle (about 5 mm3). Put the cut pieces of skeletal muscle into a petri dish and pour soda enough to cover the slices of meat. Let stand for 30 minutes to one hour and observe for any worms using a dissecting microscope. Record all observations. b. Strongyloides ratti S. ratti can be recovered from the small intestine of the dissected rat. Separate the parts of the small intestine from the cecum and large intestine and put in individual dishes of saline solution. Then slit each segment and spread open in a dish. Scrutinize for S. ratti. After examination, each segment of gut is put in a jar of saline solution. Shake vigorously, after which let stand sufficiently long (i.e., about 2 hours) for the worms to settle at the bottom. After two hours, decant the supernatant fluid. Clean the samples by repeated sedimentation and decantation until they are clear. Examine the sediment, in a petri dish in good light and under a dissecting microscope for S. ratti. Count the number of worms recovered. c. Toxocara canis Examine the stool sample of a dog. Using a toothpick, get a small amount of fecal material and mount on a slide with a drop of 0.9% saline solution. Cover the preparation with a cover slip and examine under the low power and high power objectives. Search for T. canis eggs and juveniles.

4 2. Studying Preserved Ascaris suum Obtain preserved male and female specimens of Ascaris suum. Cut open the cuticle of each specimen from one end to the other end. With the use of a dissecting microscope, identify the different parts of the parasite. Identify the ovary or uterus of the female A. suum, and mount a small amount of the contents on a clean slide with a drop of 0.9% salt solution. (CAUTION: Care must be taken in handling embryonated eggs because they are able to hatch and the juveniles migrate throughout the body in humans.) 3. Studying Nematodes Using Preserved Slides a. Examine prepared slides with whole mounts of Trichuris trichiura (adult male and female), Ancylostoma caninum (adult male and female), and Capillaria (male and female). Take note of the distinguishing features of each parasite. Draw and label the distinguishable parts of each parasite. Take note of the organs that make up the following organ systems: digestive system, male and female reproductive systems, excretory system, and nervous system. b. Examine prepared slides with the hookworm rhabditiform and filariform larvae, and the Trichinella spiralis larva. Take note of distinguishing features. Draw and label parts. c. Examine the prepared slide with the ova of Trichuris, Ascaris, and hookworm. Draw and describe each ovum. Take note of the distinguishing characteristics of each ovum. d. Examine prepared slides of a cross-section of a male and female Ascaris lumbricoides. Draw and label parts. Identify the following: intestine, cuticle, hypodermis, lateral line canals, longitudinal nerve cords, muscle layer, and reproductive organs. 4. Tabulate the distinguishing characteristics of the medically important nematodes listed in pages 1-2 of this laboratory guide. Take note of the morphological differences among members of the different orders listed above. Please refer to your textbook or other Parasitology books available. Discussion Questions 1. What is the basic structure of the nematode nervous system? 2. Describe the molting pattern of nematodes. Which stage is usually infective to the definitive host? 3. Describe the heart-lung migration of Ascaris, Ancylostoma, and Toxocara. 4. What is the single characteristic by which one may identify a nematode from a human as a pinworm? 5. Define the following terms: alae, cordon, hood, epaulet, phasmid, cloaca, ventriculus, gubernaculums, ovijector, amphidelphic, microfilaria. 6. Using examples, compare and contrast visceral larva migrans and dermal larva migrans. 7. Why is there a need to study a cross section of a nematode? 8. What is the difference between a rhabditiform larva and a filariform?

5 9. Take note of the common names of the parasites listed on pages 1 and 2. References Bush, A.O., Fernandez, J.C., Esch, G.W., and Seed, J.R. 2001. Parasitism. Dailey, M.D. 1996. Essentials of Parasitology. 6th ed. Wm.C. Brown Publishers, Boston. Moore, J. 2001. An Introduction to the Invertebrates. Pechenik, J.A. 2000. Biology of the Invertebrates. 4th Ed., McGraw Hill. Roberts, L.S. and Janovy, J.Jr. 2010. Foundations of Parasitology. 8th ed.