Download Laboratory 6 nematodes

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.
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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
Egg
J1
M2
J2
M3
J3
M4
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.
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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 twothirds of the body length.
• The anterior end of the pharynx
lacks stichocytes.
• The anus is located near the tip of the tail.
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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 squareshaped 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.
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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.
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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.
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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 longlived 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 midregion 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.
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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-tomouth” 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
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mass around the anus. The perineal region then becomes itchy and infected hosts reinfect 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.
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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 handout).
 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
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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?
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Why are they hard to eradicate?  lots of reservoir hosts
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Visual id male, female, larvae encysted, muscle infected / larvae section
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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
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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
Epidermis
Dioecious
Cloaca
Vulva
Filiform
Stichosome
Stichocytes
•
•
•
•
•
•
•
•
Viviparous
Nurse cell
Bronchial escalator
Spicule
Oviparous
Caudal bursa
Buccal capsule
Microfilaria
•
•
•
•
•
Nocturnal periodicity
Filarial worms
Pinworms
Hookworms
Whipworms