Lab 5 – Trematodes Along with the Cestodes, the Trematodes belong to the Phylum Platyhelminthes. Trematodes are flatworms and generally have: o flattened bodies, o a blind alimentary tract, o suckers for attachment to their hosts, o are hermaphrodites.

The Class Trematoda historically was split into two subclasses:  Monogenea, which includes important ectoparasites of fish, and  Digenea, including species parasitic in the bile ducts, alimentary and respiratory tracts, and blood vessels of vertebrates.

It is now widely recognized that the Monogeneans are closer to the Cestodes than they are the Trematodes. As the Monogeans and the Digeneans shared many features, we will examine both in this lab.

SUBCLASS MONOGENEA The Monogeans are primarily ectoparasites of aquatic cold-blooded vertebrates, especially fish. Some species, however, are found in the bladder of turtles and frogs. All are monoecious and the life cycle is direct. They characteristically produce few eggs. In each egg a ciliated larva, the oncomiracidium, develops, hatches, and swims about to attach to a host. The entire cycle may be completed on a single host. Diagram of general monogenean. Use this generalized diagram to help you identify the structures you see on the slides. You are responsible for the identification of the male and female reproductive structures, the digestive structures, and the opisthaptor. Monogenea are recognized by a body plan that is divided into an anterior region containing reproductive and digestive organs, and a posterior region, the opisthaptor, that serves as a holdfast. Suckers occur at the anterior and posterior of the body. The opisthohaptor (haptor), at the posterior, is very well developed and may be a single adhesive structure equipped with hooks, or it may be divided into a number of individual suckers. The oral sucker, or prohaptor, is anterior. The morphology of the opisthohaptor is very important in the systematics of this group. Just look at all the cool types!

2 Dactylogyrus extensus is an economically important parasite of hatchery fish. It has large anchors on it opisthaptor and lives on the gill filaments of its host. Heavy infections cause blood loss, erosion of epithelium, and access for secondary bacterial and fungal infections. In addition, gill irritation stimulates the production of mucus, which can smother the fish.

Slide: Dactylogyrus extensus:

Entobdella hippoglossi is a large monogenean and lives on the body surface of Halibut. • The opisthaptor is circular with a wrinkled surface and bears two pairs of large hooks. • Oviduct and short uterus lead from the single ovary to a common genital pore. • Testes are paired and empty through paired sperm ducts to a common sperm duct that leads to the genital pore. • A protrusible penis is present.

Slide: Entobdella hippoglossi

3 SUBCLASS DIGENEA Digenetic trematodes comprise most of the known trematodes and include those of greatest economic importance. This group is generally referred to as the flukes and is endoparasitic in all classes of vertebrates. Flukes are typically hermaphroditic, but some members are dioecious. All have complicated life cycles involving two to four different hosts and several different larval stages, with the first ciliated stage being referred to as a miracidium. Suckers are less well developed than in the Monogenea, but typically two are present, an anterior oral sucker and a ventral sucker (the acetabulum).

4 Morphology Use this diagram to identify the following structures:

5  Organs of attachment. Note the position and relative size of the oral sucker and acetabulum.  Spines are present in the tegument of the body surface. In some specimens the tegumental spines may have been lost during fixation.  Digestive system. Identify the mouth, pharynx, esophagus, and intestinal ceca or crura extending to the posterior end of the body.  Excretory system. Find the excretory pore at the posterior end of the body. The excretory bladder may be seen in some preparations. Flame cells and ducts DO NOT appear in fixed specimens, and are best studied in living organisms.  Reproductive system. o Genital pore. This pore shows more clearly in other digeneans. o Male reproductive system. . Testes: Two large ovoid structures in the broad portion of the fluke. . Vasa efferentia. These ducts lead from each testis to their junction with the vas deferens. They may be difficult to see. . Vas deferens. This is a single tube (sperm duct) leading to the cirrus pouch. . Cirrus pouch. A muscular pouch containing: • Seminal vesicle, a dilated portion of the tube used for sperm storage. • Cirrus, a continuation of the male duct that is protrusible. o Female reproductive system. . Ovary. A many-lobed structure that is anterior to the testes. . Oviduct. A small duct that arises from the central mass of the ovary (may not be visible). . Mehlis gland. This is a diffuse mass of gland cells near the ovary and surrounding the ootype. . Ootype. This is a dilation of the female duct between the ovary and the uterus where eggshell formation occurs. . Uterus. This is the remainder of the female duct from the ootype to the genital pore. A terminal muscular portion, the metraterm, may be seen near the genital pore. This region is difficult to see unless eggs are present. . Vitellaria (vitelline or yolk glands). These appear as grape-like clusters of follicles in the lateral regions of the body. . Vitelline ducts. These ducts lead from the vitellaria to the ootype and are difficult to see unless they contain vitelline cells. . Seminal receptacle. This is a small, thin-walled sac connected to the oviduct by a slender passage. This is lacking in some flukes. . Laurer’s canal. This is a duct of varying width arising from the seminal receptacle and leading to the dorsal surface of the body (often difficult to see).

6 Larval Stages of Digenetic Trematodes You are responsible for the identification of each of the larval stages as well as how development occurs through the larval stages. Do not learn the detailed morphology. 1. Miracidia: is a ciliated non-feeding stage of the parasite. It develops in the egg and usually hatches in the external environment. The cilia help it locate and penetrate into the appropriate mollusk intermediate. Note the eyespots.

2. Sporocyst: the metamorphosis of miricidia to sporocyst involves extensive changes. Sporocysts essentially become germinal sacs; they contain no ciliated epithelial cells, no gut and no mouth and therefore must absorb nutrients from the host. The sporocyst can develop into daughter sporocysts, rediae or in some species directly into cecaria. 3. Rediae: burst their way out of the sporocyst and migrate to the gonad of the molluscan host. They have a rudimentary but functional digestive system consisting of a pharynx, and short gut. The embryos inside the redia develop into daughter rediae or into the next stage called cercariae. 4. Cercariae: represents the juvenile stage of the vertebrate inhabiting adult. There are many varieties of cercariae and most have specializations that allow them survive for brief periods in a free-living state to make themselves available to the definitive or intermediate host. You should be familiar with the different cecarial forms and a few of the structures in them. Note the oral sucker and the acetabulum. Live material (if available): Live cecariae have been supplied for today’s lab. 5. Metacercariae is the stage between the cecaria and the adult. Metacecariae are usually encysted and can be found on the intermediate host or on aquatic vegetation, sticks or in the free-living water. The metacecaria goes into a quiescent stage and remains in readiness to excyst on reaching the definitive host.

7 Digenetic flukes are commonly separated on the basis of their general body configuration. This is done simply for convenience and does not represent any taxonomic scheme or phylogenetic relationship. The following body types are recognized: a) gasterostome - oral sucker in middle of body b) monostome - no acetabulum present c) amphistome - acetabulum at posterior extremity d) distome - “normal”, acetabulum never at posterior e) echinostome - collar of spines around oral sucker f) strigeoid - body divided into forebody and hindbody g) schistosome - blood flukes, usually dioecious

Amphistome Distome Echinostome Monostome

These have large fleshy These are the most Similar to the In these there is either bodies, with a common type, with the distomes, except that only one sucker present prominent sucker at the mouth surrounded by the oral sucker (usually only the oral posterior of the body the oral sucker and a surrounded by a sucker), or there are two (e.g. Gastrodiscoides ventral sucker, present prominent collar, suckers, but one very hominis ) anywhere on the ventral equipped with spines reduced, or in some cases surface except the (e.g. Echinostoma no suckers) extreme posterior sp.) (e.g. Notocotylus (e.g. Fasciola hepatica) attenuates)

Family Dicrocoeliidae These flukes are parasites in the liver, gall bladder, bile duct, and pancreatic duct of reptiles, birds, and mammals. Features include:

 Suckers are well developed.  Cirrus pouch anterior to the acetabulum.  Ovary posterior to the testes.  The uterus has ascending and descending limbs.

8 Dicrocoelium dendriticum is a species that inhabits the bile duct of sheep. Its life cycle is one of the most interesting found in the Trematoda. Eggs in the feces of the host are eaten by land snails and hatch in the intestine, where miracidia penetrate the gut wall. Mother sporocysts produce daughter sporocysts in which cercariae develop. Cercariae leave the daughter sporocysts and accumulate in the mantle chamber where mucus secreted from the snail envelops them to form “slime-balls”, which are deposited on the vegetation as the snail moves about. Slime-balls are eaten by ants and metacercariae encyst in the body cavity. A few however, migrate to the subesophageal ganglion and encyst there, altering the ants behavior so that when the temperature drops in the evening, infected ants climb to the tips of grasses and hang there by their mandibles, thus allowing the definitive host to more easily ingest them while grazing.

Slide: Dicrocelium dendriticum: note the tandem to oblique testes anterior to the ovary, location of the vitellaria, and general body shape. Use the diagram in the handout to identify as many structures as possible.

9

Family Opisthorchiidae Flukes in this family inhabit the liver, bile ducts, and gall bladder of fish-eating reptiles, birds, and mammals. Fish and sometimes amphibians serve as second intermediate hosts.  Suckers are weakly developed and the testes are posterior and tandem.  Ovary is pretesticular and the genital pore is located just anterior to the acetabulum.  Vitelline follicles are large, lateral, and usually anterior to the gonadal region. Clonorchis sinensis (the Chinese liver fluke) does not occur in North America, but is an important parasite of humans, cats, and dogs in Asia.

Slide: Clonorchis sinensis: Study this species noting the branched, tandem testes, small ovary, enlarged seminal receptacle, and Lauer’s canal.

10 Family Echinostomatidae These are spinous-bodied flukes with a head collar, which surrounds the oral sucker and contains a single or double row of large spines. Ventral sucker is larger than the oral sucker, and vitellary are well developed and lateral. A seminal receptacle is absent. They are intestinal parasites of reptiles, birds, and mammals. Echinostoma revolutum is one of the most common trematodes of wild birds and mammals. This species shows little specificity for its intermediate or definitive hosts. Sporocysts and two generations of rediae are produced in many different species of snails. More than 20 species of birds and nine species of mammals have been reported as hosts. Slide: Echinostoma revolutum: Study this slide noting the head collar and reproductive structures. A diagram of Echinostoma revolutum has been included in your handout.

Family Fasciolidae

Fasciolid trematodes are  Very large, leaflike flukes of mammals.  Suckers are close together and near the anterior of the body.  Testes are multibranched and tandem.  The ovary is also branched and is pretesticular in location.  Cirrus and cirrus pouch are well developed.  The genital pore is anterior to the acetabulum.  The uterus is short and entirely anterior to the ovary.  Vitelline follicles are very small, abundant, and distributed throughout the body.

Fasciola hepatica was the first trematode described, and the first trematode whose life cycle was elucidated. The sheep liver fluke is a parasite of the liver and bile duct of sheep and cattle throughout the world.

11

Slide: Fasciola hepatica Study your slide and note the branched testes, ovary, gut, suckers, uterus, vitelline follicles, and cirrus pouch.

12 Fasciolopsis buski is the giant intestinal fluke of humans and swine in Asia.

Slide: Fasciolopsis buski: Note the unbranched gut, the large ventral sucker, and the lack of a cephalic cone.

Family Schistosomatidae Schistosomes are parasites of the blood vessels of birds and mammals. Several members of this family are very important economically as parasites of the circulatory system of humans and domestic animals.  Adults are dioecious, with a distinct sexual dimorphism.  Females are usually more slender and longer than males, and are frequently located in a longitudinal groove (gynaecophoric canal) of the latter.  Suckers are poorly developed and a pharynx is lacking.  The intestinal ceca are fused in the posterior of the body to form a common cecum.  Testes number four or more and the ovary is ovoid or spirally coiled.  Vitelline follicles are numerous and the uterus is short.  Only sporocysts and cercariae are present in the life cycle, with cercariae directly penetrating the definitive host.

13 Schistosoma mansoni is a parasite of the mesenteric veins surrounding the intestine of humans in Africa, South America, and Puerto Rico.

Slide: S. mansoni male and female in copula.

Slide: S. mansoni male. Study these slides noting the oral sucker, acetabulum, gynaecophoric canal, and 6-9 testes in the male, Slide: S. mansoni female. Note the ovary in the anterior half of the female. Note the absence of a pharynx and the characteristics of the gut.

14

Slides: Eggs of S. mansoni have a prominent lateral spine, which is diagnostic for this species. Eggs of S. haematobium have a prominent spine at the end of the egg. Eggs of S. japonicum have a very small spine that may not be visible without very close examination of the egg.

Slide: Tissue section of S. mansoni infected liver.

Slide: Tissue section of S. mansoni infected intestine.

Other Schistosomes: Schistosoma haematobium is the causitive agent for urinary schistosomiasis, especially in Egypt, other parts of Africa, India, and the Middle East. Gravid worms live in pelvic veins primarily, and eggs are voided with the urine. Schistosoma japonicum is the third schistosome of humans and is limited to the Far East where it causes rectal schistosomiasis. The egg of this species is ovoid to round and contains a rudimentary lateral spine. Schistosomatium douthitti is one of the few mammalian schistosomes in North America. It is a parasite of the mesenteric veins and liver of voles and muskrats. This is one of the species whose cercariae are often responsible for “swimmer’s itch” or “schistosome dermatitis” in humans.

15

Family Troglotrematidae These are fleshy flukes that parasitize the lungs, nasal cavities and intestines of many birds and mammals. Adults frequently occur together in pairs within cysts.

 The body is spinose.  Suckers well developed.  Ovary prestesticular.  The genital pore is located near the acetabulum.

Paragonimus westermani is a parasite that is restricted to the Far East and parasitizes feline, canine, and porcine mammals as well as humans. Eggs of this fluke hatch in water and the miracidia penetrate snails where sporocysts, mother rediae, and daughter rediae are produced. Cercariae leave the snail and encyst in crabs and crayfish. Humans become infected by ingesting raw or partially cooked crabs or crayfish. Adults have been known to survive for up to 20 years in the lungs of humans.

Slide: P. westermani adult

16

Slide: P. westermani egg

17

Learning Objectives

1 .Know general characteristics - Compare monogenea and digenea 2. Monogenea - One host simple life cycle - Host, life cycle stages, anatomy - Dactylogyrus: host, host tissue, economic importance, visual id 3. Digenea - 2+ hosts, complicated life cycle, 1st intermediate: mollusc - General morphology and function: reproductive organs vary and can be used for identification - Larval stages: anatomy, form and function, hosts, visual id 4. Family Dicrocoeliidae (liver flukes) - Dicrocoelium dendriticum - General characteristics - Tissue in definitive host - Life cycle - How does this species alter its intermediate host behavior to increase chance of transmission - Visual id 5. Family Opisthorchiidae (liver flukes) - Chlonorchis sinensis - General characteristics - Life cycle - Tissue infected - Visual id 6. Family Echinostomatidae (intestinal flukes) - Know that they are spinous - Echinostoma revolutum slide - Hosts: low host specificity - Tissues infected - Visual id 7. Family Fasciolidae - General characteristics as they apply to the two species - Visual id, life cycles, compare and contrast F. hepatica and F. buski 8. Family Schistosomatidae (blood flukes) - Dioecious with sexual dimorphism - General characteristics - Schistosoma mansoni: tissue in definitive host - Visual id: male, female, in copula - find eggs in intestine and liver - Know that these eggs cause the pathology - Life cycle - Other examples of Schistosomes, diagnostic feature (eggs) and pathologies 9. Family Troglotrematidae (lung flukes) - Tissue infected in definitive host, visual id , life cycle, host, Paragonimus westermani

Vocabulary

• Monoecious • Prohaptor • Sporocyst • Dioecious • Acetabulum • Rediae • Oncomiracidium • Operculum • Cercariae • Opisthaptor • Miracidia

18

19