Lab 6 - PHYLUM NEMATODA Nematodes are almost unbelievably abundant. Not only are there more than 15,000 known species of roundworms, but there are many thousands of individual nematodes in even a single handful of garden soil. Some species of roundworm may contain more than 27 million eggs at one time and lay more than 200,000 of them in a single day. Some scientists have estimated that there may be as many as half a million unknown species of roundworm yet to be discovered; an estimate based on the fact that many new species are still being discovered, that relatively few people are looking for more species, and that most roundworms look pretty much alike. If the estimated number of species is anywhere close to correct, it would mean that roundworms are the second most diverse group of animals, trailing behind only the arthropods.

General Characteristics Nematodes are bilaterally symmetrical, worm-like organisms that are surrounded by a strong, flexible noncellular layer called a cuticle. The body plan is simple. The body of a nematode is long and narrow, resembling a tiny thread in many cases, and this is the origin of the group's name. The word "nematode" comes from a Greek word “nema” meaning "thread". The epidermis (skin) of a nematode is highly unusual; it is not composed of cells like other animals, but instead is a mass of cellular material and nuclei without separate membranes. This epidermis secretes a thick outer cuticle, which is both tough and flexible. Most are dioecious and show considerable sexual dimorphism, with the female usually larger and the tail of the male being more curled. In females, the reproductive tract opens separately from the digestive tract, while in males the digestive tract joins the reproductive tract forming a cloaca. Nematodes vary in size from less than 1 mm to over 1 meter in length. Juvenile nematodes require several molts before the adult stage is reached, and development may be either direct or may require an intermediate host. 2

Development All nematodes pass through 5 developmental stages separated by 4 molts. Pre-adult stages are referred to as larval or juvenile stages and differ from the adult stage in both size and in the extent of development of the reproductive system.

M1 M2 M3 M4

Egg J1 J2 J3 J4 Adult

Nematode Morphology Digestive System: The nematodes have a complete digestive tract. Find the mouth opening at the blunt end of the worm. The mouth opens into the characteristic pharynx. In most cases the pharynx is highly muscular -- it often has a terminal bulb where it joins the intestine. A nerve ring encircles a constriction of the pharynx, but it is seldom visible. The pharynx is continuous with the thin-walled intestine (which lacks any musculature). The digestive system terminates near the end of the worm at the anus. (In male nematodes the reproductive and digestive system join together forming a cloaca, which terminates at the anus.) Reproduction: Most female nematodes have a complex, double set of reproductive organs. In nematodes the reproductive system is in the form of a continuous tube, which is usually subdivided into ovary, oviduct, and uterus. The ovaries are separated from the double uteri by an oviduct in which fertilization typically occurs. Uteri flow into a common vagina that serves as a canal for expulsion of eggs and for movement of sperm upwards towards the oviducts. The vagina opens to the outside at the genital pore (also called the vulva). The uterus can be distinguished as that part of the tract in which young worms (larvae) can be seen moving about. Most female nematodes lay eggs, but in some like the vinegar eel, the trichina worm, and filarial worms, the eggs hatch before they leave the uterus. The adult males are distinctly smaller than the females. This is the case with most nematodes. They can be differentiated from females by their smaller size, by the presence of chitinous spicules, the accessory organ of copulation, near the cloaca, and by the lack of a genital pore (other than that of the anus). The spicule serves to guide sperm into the female tract during copulation. Male nematodes commonly have a single reproductive tract. The vas deferens (sperm duct) joins the digestive tract to form the cloaca, which leads to the anus. An enlarged portion of the sperm duct, the seminal vesicle, is often present anteriorly. The vas deferens is continuous anteriorly with the testis -- it is often difficult or impossible to differentiate these portions of the system. We have included a diagram of general nematode morphology in this hand out. Use this diagram to become familiar with the nematode. 3

Schematic of Nematode Female

Schematic of Nematode male

Schematic of

Nematode Male

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CLASS ENOPLEA: Order Trichurida

Nematodes in this Order are characterized by: • A body that is filiform anteriorly. • A mouth without lips. • An extremely long, thin, nonmuscular pharynx, called a stichosome. Within the stichosome is a row (or rows) of large cells, called stichocytes. • A single reproductive system in females, and the majority of species are oviparous. The vulva is commonly located near the base of the pharynx. • Males have a single spicule or none at all. • The life cycle is usually direct, with no intermediate host required. • Parasites of nearly all organs of all classes of vertebrates.

You are responsible for two nematodes in the Order Trichurida; Trichuris trichiura and Trichinella spiralis. Trichuris tricuhura, the human whipworm, is a human parasite found throughout the world, but most prevalent in the tropics. The incidence of infection may reach 25% in parts of the southeastern United States. Typically, infections involve fewer than 100 worms and are relatively symptomless. However, in more intense infections, the mortality rate can be 1 in 1000. The worms are relatively large (30-50mm), with an extremely narrow anterior end and broader posterior ends, hence the name “whipworm”. These parasites occur in the large intestine where they embed their anterior ends beneath the mucosal surface and feed on cells in the lamina propria. The protruding posterior ends of the worms find each other for mating and females deposit eggs into the intestinal lumen so they pass out with the feces. Slide: Adult Trichuris trichiura, male and female (w.m.) Study the adult worm: • Distinguish the long filiform anterior end from the fusiform posterior. • Note the single row of large cells (stichocytes) surrounding the long, thin pharynx. The entire structure is referred to as the stichosome. Stichocytes secrete material that aid in digestion and that modulate the host reaction to the parasite. • The pharynx occupies about two- thirds of the body length. • The anterior end of the pharynx lacks stichocytes. • The anus is located near the tip of the tail. 5

In the male: • Note the coiled tail • The single spicule surrounded by a spiny spicule sheath. • The testis is singular, long, and convoluted, and gives the appearance of square- shaped compartments along its length. • Follow the testis forward from the region of the cloaca to a point near the termination of the stichosome. The tube now turns back on itself as a large uncoiled vas deferens. • Posterior, the vas deferens narrows, then widens again to form an ejaculatory duct that joins the intestine to form the cloaca. In the female • Note the bluntly rounded posterior end. • The vulva is located near the anterior end of the fusiform body region, near the junction of the pharynx and intestine. • A coiled vagina runs to the posterior from the vulva to its junction with a wider uterus. • The uterus runs to a coiled oviduct and sacculate ovary. • Eggs within the uterus are unembryonated, and have a characteristic barrel shape with a plug at each end. Slide: Trichuris eggs: Trichuris eggs are easily recognized by their prominent bipolar plugs and large size. Females produce 3000 to 20,000 eggs per day. Eggs embryonate within three weeks after leaving the host’s body and can remain infective for months if they are deposited in moist soil in the shade. Infection is acquired when a suitable host ingests embryonated eggs. Adults live for several years, so large numbers may accumulate in a person, even where the rate of new infection is low. 6

Trichinella spiralis, the trichina worm, is one of the most studied of all nematodes. It is the smallest nematode parasitic in humans, has one of the most unique life cycles, is one of the most widespread, and is one of the most medically important parasites in the world. Adult worms lie buried in the mucosa of the small intestine. Males die shortly after copulation. Females are viviparous, giving birth to living young in the tissues of the intestine. Juvenile nematodes are transported via the lymph or blood to all parts of the body. Further development only occurs in striated muscle, especially those muscles that are active. They penetrate individual muscle fibers, absorb nutrients from the muscle cell, and increase in length to about 1.0 mm in eight weeks, at which time they are infective. During this time they assume a spiral shape and become encysted by infiltrating leukocytes. They may remain viable for many years. Transmission is through ingestion of the larvae infected meat. Upon ingestion by a host, the cycle repeats; therefore, one animal serves as both definitive and intermediate host, with the juvenile and adult inhabiting different organs. Most mammals are susceptible to infection. Trichnella spp. generally have reduced host specificity, although some species tend to occur in different host associations. The life cycle depends on scavenging food chains: first stage larvae embedded in muscle ingested by a predator or scavenger and develop to adulthood and produce infective larvae in the muscles of the scavenger. Humans usually acquire infections through eating undercooked pork. Pigs maintain infections because agricultural practices often facilitate transmission (feeding of offal to pigs). See attached life cycle. 7

Slide – Trichinella spiralis, adult male and female The females are twice as large (about 3.0 mm) as the males (about 1.5 mm), and are therefore quite easy to separate. In both sexes note the stichosome type of pharynx. In the male: • The greatly enlarged seminal vesicle should be evident beginning just posterior to the junction of the pharynx and intestine. Also note the two large genital papillae. • A spicule is absent. On a female: • Locate the vulva that opens in the anterior third of the body. Slide – Trichinella spiralis encysted: Look at encysted juveniles of T. spiralis in tissue section. Larvae penetrate individual striated muscle cells and will provoke formation of a nurse cell with stichosomal secretions. The nurse cell nurtures and protects the parasite inside the cell. Note the characteristic shape the juvenile assumes, and observe the nature of the cyst wall.

Slide: Trichinella spiralis larvae sec See entire larvae in a muscle cell. 8

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CLASS: RHABDITEA

Order: Ascaridida Nematodes in this Order:  Mostly large, stout parasites of vertebrates.  The mouth is surrounded by three conspicuous lips.  A buccal capsule and pharyngeal bulb is absent in most species.  Males have two copulatory spicules of equal or unequal length, and a pointed, ventrally coiled tail.  Females are oviparous, possess a double reproductive system, and have a blunt tail.  Eggs are thick-shelled and require a long period of incubation before they become infective.  Development is usually direct.

Ascaris lumbricoides is one of the largest and most common parasites found in humans. The adult females of this species can measure up to half a meter long (males are generally shorter), and it is estimated that 25% of the world's population is infected with this nematode.

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The adult worms live in the small intestine and eggs are passed in the feces. About two weeks after passage in the feces the eggs contain an infective larval or juvenile stage, and humans are infected when they ingest infective eggs. The eggs hatch in the small intestine; the juvenile penetrates the small intestine and enters the circulatory system, and eventually entering the lungs. In the lungs the juvenile worm leaves the circulatory system and enters the air passages of the lungs. The juvenile worm then migrates up the air passages into the pharynx where it is swallowed, and once in the small intestine the juvenile grows into an adult worm. This process is called the bronchial escalator. Ascaris infections in humans can cause significant pathology. The migration of the larvae through the lungs causes the blood vessels of the lungs to hemorrhage, and there is an inflammatory response accompanied by edema. The resulting accumulation of fluids in the lungs results in "ascaris pneumonia," and can be fatal. Heavy infections can obstruct the bowel and lead to perforation. Display: Ascaris whole worms. Note the lateral lines that appear as paired white cords along the length of the body. You may be able to find the vulva of the female that opens about one third of the way from the anterior end.

Slide: Ascaris eggs: a single female can produce up to 200,000 eggs each day! The eggs have a characteristic convoluted outer shell; they are long- lived and persist in the soil for more than a year.

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Slide: Ascaris cross section female and male: Nematode Histology and Organology You will need to know the histology of a nematode as seen in cross section. Use the Ascaris cross sections to become familiar with the insides of a nematode.

Female Male

We have included a diagram above to help you identify the following structures:

1. Cuticle. A multilayered, non-living, non-cellular outer region of the body, and is secreted by the underlying hypodermis. 2. Hypodermis. Lies just beneath the cuticle and is usually syncytial in adult worms. The nuclei lie in four thickened portions that project into the pseudocoel. These hypodermal cords are longitudinal and divide the body musculature into four distinct groups. There are two lateral, one dorsal, and one ventral cord. The dorsal and ventral cords contain longitudinal nerve trunks, and the lateral cords contain the lateral canals of the excretory system in most species. One of the primary functions of the hypodermis is the secretion of the cuticle. 3. Muscle. All body wall muscles are longitudinal. Note that the muscle cells consist of an inner, noncontractile (sarcoplasmic) portion containing the nucleus, and an outer contractile (fibrillar) portion. 4. Nerves. The major nerve cords may be seen in the dorsal and ventral cords of the hypodermis. These are connected to two main concentrations of nerve elements, one in the pharyngeal region and one in the anal area. 5. Digestive system. Only the intestine will be seen in these sections, made in the mid- region of the body. The intestine is usually collapsed. Note that only a thin basement membrane separates the intestine from the pseudocoel. Also note the single layer of epithelial cells that make up the bulk of the intestine wall. 12

6. Reproductive system. Because the reproductive system of Ascaris is double and greatly coiled, many portions of the same organ may be seen in one section. In the female the ovary is characterized by being largely solid, with a small central core. Divisions between the cells appear similar to the spokes of a wheel. The oviduct resembles the ovary, but has a lumen. The two uteri, which should be cut through only once, are larger and contain developing eggs. In cross section of a male, the testes, vas deferens, and seminal vesicle may be present. Cells within the testes are small and compactly grouped. Those of the vas deferens are larger and not so compactly arranged. The seminal vesicle is large, single, and may contain ameboid sperm.

Order: Oxyurida

Oxyuroid nematodes:

 Are small pin-shaped parasites of vertebrates and invertebrates.  Are called pinworms because females have characteristically long, pointed tails.  Have a small buccal capsule leading to a pharynx with a well-developed end bulb (=oxyuroid type of pharynx).  Males have one or two spicules of equal length.  They are usually parasites of the cecum or large intestine of their hosts.  No intermediate hosts are required in the life cycle.

Pinworms are common in mammals, birds, reptiles, and amphibians but are rare in fish. Most domestic birds and mammals harbour pinworms however, cats and dogs do not. Insects and millipedes commonly are infected. Enterobius vermicularis may be the most common nematode parasite of humans. Children are most commonly infected, with prevalence rates above 50% being reported in the early school-age children in some areas of the United States. The life cycle is direct; therefore, “hand-to- mouth” transmission is common. Worms occur in the intestine. Females mate and accumulate eggs in their uterus. When ready to lay their eggs, the female will migrate out of the anus and deposit the eggs in a 13 mass around the anus. The perineal region then becomes itchy and infected hosts re- infect themselves when they scratch and later put their fingers in their mouths. Needless to say pinworms tend to be a larger problem with young children who are unable to curb their habits in this regard! Effective non-toxic treatments exist but re-infections are common in those behaviourally disposed to transmission. Consult your text and lecture notes for a detailed account of the epidemiology and control of human pinworms.

Slide: Enterobius vermicularis, the human pinworm. Both a male and female worm are available for study. Note the:  General fusiform body shape.  The well-developed end bulb of the pharynx.  The long pointed tail and the position of the vulva on the female.  Cervical alae are conspicuous on both sexes, but the intestine in the female may be obscured by the gravid uterus filled with eggs.  The ovaries degenerate in gravid females.  The female is larger than the male.  The curved posterior end of the male, and the single, simple spicule.

Order: Strongylida This order contains the bursate nematodes characterized by the presence (in males) of a caudal bursa, a modification of the posterior end that aids males in handling the females during copulation. Most species are parasites of the vertebrate intestine and have a direct life cycle, requiring no intermediate host. Super family: Ancylostomatoidea This superfamily consists of the so-called hookworms; the name being derived from the characteristic hook-like shape of the adults. This group of nematodes:  Has heavily sclerotized mouthparts, i.e. cutting plates or teeth within the buccal capsule.  They are all blood-feeders that inhabit the upper intestine of mammals, and occasionally amphibians and reptiles. 14

The life cycle of hookworms is direct, requiring no intermediate hosts. Females in the small intestine lay unembryonated eggs that develop into the 2-4, or several-cell stage by the time they leave the host with the feces. Embryonation is completed in one to several days outside of the host. First stage juveniles are rhabditiform (=pharynx with a characteristic constriction at the level of the nerve ring) and live in the feces. The first molt occurs in 2-3 days, and the second in another 5 days. The third stage is a filariform juvenile, and this is the infective stage. Infection of the definitive host is by direct penetration. The pre-patent period is approximately five weeks. See attached life cycles.

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Ancylostoma caninum is the common hookworm of dogs, cats, fox, and other carnivores in both temperate and tropical or subtropical areas of the world Slide – Ancylostoma caninum, adult (male and female): Study this specimen and note:  The two sets of teeth in the buccal capsule. Each set consists of three individual teeth (see hand- out).  The club-shaped pharynx that is present in all hookworms. The pharynx is very muscular, corresponding to its action as a powerful pump.  On a male worm study the conspicuous copulatory bursa consisting of three lobes and supporting fleshy rays. Contrast this with the simple, conical tail in the female.

Slide: Ancylostoma caninum, in copula

Necator americanus, the “new world hookworm,” is a smaller species than Ancylostoma, and is found throughout much of the world. It is very common in parts of the southeastern United States. Slide –Necator americanus adult (male and female): Note that:  Teeth are absent in this species, being replaced by a pair of cutting plates.  The anterior end is characteristically curved in a direction opposite from that of the rest of the body.  The copulatory bursa is much smaller than that of the male Ancylostoma. Slide - Necator americanus eggs.

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Order: Spirurida This is the most diverse of nematode orders and includes several important pathogens of humans. Life cycles are always indirect and use a variety of arthropods as intermediate hosts. Typically, eggs passed in the feces are ingested by the intermediate host; larvae hatch from the egg and penetrate into the body cavity where they develop to the third larval stage. When a suitable vertebrate host ingests the intermediate host containing the infective L3 stage: the worms migrate to their final site in the host and develop to adulthood; completing the life cycle.

Superfamily: Filarioidea The so-called filarial worms include several of the most important pathogens of humans and domestic animals. Filarioid nematodes are: long, thread-like parasites that inhabit the tissues, body cavities, blood and lymphatics of their hosts. They are commonly referred to as filarial worms. Females are usually ovoviviparous, giving birth to living young known as microfilariae. The life cycle is indirect. Many species of arthropods function as vectors of filairoid nematodes.

Dirofilaria immitis, the dog heartworm, is a common parasite of canids and occasionally felids throughout much of the world. The adults are large nematodes that inhabit the right ventricle and pulmonary arteries where the female deposits unsheathed microfilariae into the bloodstream. Microfilariae exhibit a nocturnal periodicity, being more abundant in the peripheral blood during the night than during daylight. Well over 60 species of mosquitoes have been incriminated as potential vectors of D. immitis, but natural vectors are primarily species in the genus Aedes. Microfilariae invade the malpighian tubules of the mosquito and develop to the third stage juvenile. This stage breaks out of the tubules and 17 migrates to the labium. When the mosquito takes a blood meal, the juveniles break out of the labium and enter the host via the puncture made by the mosquito.

Slide: Unsheathed microfilaria of Dirofilaria immitis

Onchocerca volvulus is a human parasite that inhabits the subcutaneous connective tissues and is the causative agent of river blindness. Worms are transmitted by black flies (development from L1-L3 occurs in the black flies). These flies have aquatic larvae that need fast flowing streams of well-oxygenated water to develop and this requirement explains the more local distribution of the worms. Adults worms occur just beneath the skin in humans where they become surrounded by a tumor like growth of connective tissue. Females Onchocera release unsheathed microfilaria that accumulate in the skin and are picked up by feeding flies in the family Simuliidae. Pathology is associated with chronic infection and arises largely from microfilariae that block local blood flow and elicit chronic inflammation. The eye can act as a trap for microfilariae and chronic host reaction to the presence of microfilariae eventually leads to blindness.

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Slide - Tissue cross-section of a section of an Onchocerca nodule. Note the microfilariae throughout the section. Onchocerca microfilariae can often be distinguished from Wuchereria by examining the nuclei in the tail worm. In Onchocerca, the nuclei reach the tip of the tail. In Wuchereria, the nuclei DO NOT reach the tail tip. It will probably be difficult for you to see these nuclei with the microscopes in our lab.

Wuchereria bancrofti: Adult worms occur in the lymphatics, particularly of the groin and lower extremities. Once male and female nematodes mate, the female ovoviviparously produces microfilariae (L1), which then move through the circulatory system and collect in the lympathics during the day and emerge at night when night biting mosquitoes are most active. Once ingested by a mosquito, the microfilariae penetrate the insects’ gut wall and move to the thoracic muscles where they mature (through two life stages) into third-stage infective larvae (L3) Pathology varies greatly with signs of inflammation ranging from mild to severe. Elephantiasis characterizes a disease associated with severe inflammation. The disease is caused by a chronic blockage of lymphatics, not by adults, but by microfilariae that get trapped in the lymphatics and die. Microfilariae elicit a strong chronic response from macrophages leading to connective tissue build-up, improper drainage of effected tissues and pressure atrophy of swollen tissues.

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Slide – Wuchereria bancrofti microfilaria

Learning Objectives

1. Know general characteristics - Know what is special about their cuticle + epidermis - Know development + morphology 2. Order Trichurida - Know general characteristics for id + anatomy 3. Trichuris trichura - Human whipworm - Epidemiology, anatomy - Purpose of thick and thin end - Visual id males, females, eggs - Embryonation, infectivity 4. Trichinella spiralis - (adult slide not good) - Largest intracellular parasite - Life cycle, tissue + transmission - Females are viviparous - What is special about larvae? - Why are they hard to eradicate?  lots of reservoir hosts - Visual id male, female, larvae encysted, muscle infected / larvae section 20

5. Order Ascarida - General characteristics 6. Ascaris lumbricoides - Epidemiology, pathology - Size - Life cycle, transmission, bronchial escalator - Eggs, embryonated or unembryonated, number of eggs (thick outer shell, persistence) - Cross section: id parts+ sex 7. Order Oxyurida - Know general charactersitics 8. Enterobius vermicularis - Pinworm - Most common nematode parasite of human - Life cycle, transmission - Itchy bum! - Visual id female, male 9. Order Strongylida 10. Superfamily Ancylostomatoidae - Hookworms - General characteristics - Life cycle 11. Ancylostoma caninum - Where? - Host, mouthparts – buccal capsule, caudal bursa (copulatory bursa) - Visual id in copula, male, female 12. Necator americanus - Where? - Visual ID male, female, eggs - Mouthparts – caudal bursa 13. Order Spirurida - General characteristics 21

14. Superfamily Filarioidae - Filarial worm - General characteristics 15. Onchocerca volvulus - Life cycle, pathology - Vector - Visual id nodule cross-section containing adults containing microfilariae 16. Wuchereria bancrofti - Life cycle, pathology, disease, transmission - Visual id mircofilariae

Vocabulary

• Cuticle • Viviparous • Nocturnal periodicity • Epidermis • Nurse cell • Filarial worms • Dioecious • Bronchial escalator • Pinworms • Cloaca • Spicule • Hookworms • Vulva • Oviparous • Whipworms • Filiform • Caudal bursa • Stichosome • Buccal capsule • Stichocytes • Microfilaria