Download PSEUDOCOELOMATE LABORATORY Phylum Gastrotricha 1

PSEUDOCOELOMATE LABORATORY
Phylum Gastrotricha
1. Lepidodermella squamata – live specimens, observe location
Phylum Rotifera
1. Rotifer – prepared slide
2. Rotifer – living specimens, observe locomotion, feeding and behavior
Phylum Acanthocephala
1. Acanthocephalus jacksoni - prepared whole mount slides
Phylum Nematoda
1. Ascaris lumbricoides
a) whole preserved specimens for dissection
b) cross section slides of internal anatomy
2. Trichinella spiralis (pork worm)
a) slides of parasites in muscle tissue
3. Ancylostoma caninum (hookworm)
a) slides
4. Enterobius vermicularis (human pinworm)
Phylum Ectoprocta
1. preserved and dried ectoprocts for observation
2. slides for internal anatomy
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Group Pseudocoelomates
The pseudocoelomates are a diverse group of organisms that are grouped together in the phylum
ASCHELMINTHES by some authors. In our work, however, we will consider each pseudocoelomate
group to represent a separate phylum instead of classes within the phylum Aschelminthes. Using this
approach, the terms pseudocoelomate and aschelminthes are descriptive terms with no taxonomic
meaning. The pseudocoelomate phyla are Nematoda (round worms), Rotifera (wheel animalcules),
Gastroticha, Kinorhyncha,, Nematomorpha (horsehair worms) and ectoprocts.
A number of common characteristics among these groups justifies considering them together. The
pseudocoelom is one important characteristic shared by all these groups. The pseudocoel is a space
between the intestine and body wall which is embryonically derived from the blastocoel and is filled with
fluid and the reproductive and excretory systems. It is believed that the pseudocoelom represents an
evolutionary stage between the acoelomates, in which the body cavity is filled with undifferentiated
mesodermal cells, and the coelomates, in which the body cavity is completely lined with mesoderm.
What are the advantages of a coelom?
A second major difference between the pseudocoelomate phyla and the phyla we have considered
thus far is the presence of a complete digestive system: mouth => intestine=> anus. Cellular
specialization in this system allows ingested food to be processed along a one-way passage, with
unassimilated matter being ejected out the anus, rather than having it returned and passed back out the
oral opening as in cnidarians and most platyhelminthes. The advantage of a one-way digestive system
should be obvious.
Pseudocoelomates also possess a hardened cuticle. The cuticle is not only an extraordinarily
effective protection against harmful external substances (or digestive enzymes in the case of internal
parasites), but also serves as an exoskeleton, allowing an extensive area for muscle attachment and
support.
Still another remarkable characteristic of pseudocoelomates is a tendency towards constancy of
cell number or eutely, in which the organs and often the entire organism are composed of a precise and
relatively small number of cells. The number may be constant not only for a species, but also for larger
taxonomic groups. The nematode Ascaris, for example, as well as other closely related genera have a
nervous system consisting of precisely 162 cells.
In today's laboratory, we will examine the functional morphology and biology of some
representative nematodes and rotifers and also try to locate them in their natural habitat.
PHYLUM GASTROTRICHA
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PHYLUM ROTIFERA
The rotifers and acanthocephalans appear to be more closely related to Lophotrochozoa, a large alliance
of protostomes that includes molluscs and annelids.
Rotifers are multicellular animals with body cavities that are partially lined by mesoderm. These
organisms have specialized organ systems and a complete digestive tract that includes both a mouth and
anus. Since these characteristics are all uniquely animal characteristics, rotifers are recognized as animals,
even though they are microscopic. Most species of rotifers are about 200 to 500 micrometers long.
However a few species, such as Rotaria neptunia may be longer than a millimeter. Rotifers are thus
multicellular creatures that make their living at the scale of unicellular protists.
Rotifers are microscopic and the majority are found in fresh water. Some rotifers are characteristic of
extremely transient freshwater habitats such as temporary puddles or mosses which hold rain water. The
body of rotifers is cylindrical in cross section, and, like that of nematodes, is covered by a cuticle. Unlike
nematodes, however, the cuticle may be divided into sections so that telescoping of the body is possible.
This cuticular segmentation is not reflected by the internal organs and is referred to as
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pseudosegmentation. The anterior end of the body is in the form of a crown of cilia called a corona
which serves as a food gathering device and functions in swimming. The posterior end of the body may
be in the form of a stalk and tapers into a foot (toes and spurs) supplied with cement glands in sessile
species, or may be relatively unmodified in free-swimming species.
Examine the structure and movements of the live rotifers. Note that rotifers do not have distinct
muscle layer, but possess muscle fiber bundles which traverse the pseudocoel and attach to the cuticle.
The feeding machinery of rotifers is unique. The anterior end of rotifers bears a flat, ciliated
surface called a corona. The coronal cilia create a current of water which draws food particles toward the
ventroanteriorly- positioned mouth. The mouth leads into a highly muscularized pharynx (mastax) that
bears cuticularized jaws called trophi. Muscles in the pharynx cause the jaws to produce a grinding and
chewing action. Salivary glands open into the pharyngeal cavity and begin the digestive process.
Posterior to the pharynx are the narrow esophagus and sac-like stomach. Paired gastric glands open into
the stomach and secrete digestive enzymes. Cilia line the digestive tract and move food through the
intestine which terminates in an anus. The digestive system of nematodes lacks cilia. Can you suggest a
reason for this difference between nematodes and rotifers?
The posterior end of a rotifer is characterized by
Observe living rotifers feeding on a drop of Congo-red-stained yeast. Describe the pattern of water
movement created by the corona and the fate of the food. Can you detect a pH change in the digestion
process?
Rotifers have flame bulbs, similar to those found in flatworms for osmoreregulation. They are
generally found as a pair lying on either side of the mastax. You may be able to see the flame bulbs if you
are patient and slow down the wheel animicules with a drop of methyl cellulose (protoslo).
Reproduction in rotifers is rather unusual as are their morphology and feeding habits. Several types of
reproduction have been observed in rotifers. Some species consist only of females that produce their
daughters from unfertilized eggs, a type of reproduction called parthenogenesis. In other words, these
parthenogenic species can develop from an unfertilized egg, asexually. Other species produce two kinds
of eggs that develop by parthenogenesis: one kind forms females and the other kind develops into
degenerate males that cannot even feed themselves (sexual dimorphism). These individuals copulate
resulting in a fertilized egg developing within the rotifer. The males survive long enough to produce
sperm that fertilize eggs, which then form resistant zygotes that can survive if the local water supply
should dry up. The eggs are released and hatch in the water. If the egg develops in the summer, the egg
may remain attached to the posterior end of the rotifer until hatching. The female reproductive system of
rotifers consists of a single ovary and a yolk-producing vitellarium. The eggs pass from the oviduct into
the cloaca and then to the outside. Male rotifers are extremely rare and unknown in some species. They
are much smaller than females, lack a digestive system, and have a single testis which discharges into the
cloaca which terminates in an armored penis. During copulation, the penis is inserted into the female
oviduct by puncturing the cuticle.
A generalized life cycle of rotifers is given in the figure at the end of this handout. There are two
types of rotifer females, amictic and mictic. Amictic female always reproduce parthenogenetically
producing eggs that all develop into females, whereas mictic females may reproduce sexually. Mictic
females produce dwarf males as well as resting eggs. The cycle begins with parthogenetic reproduction
which builds the populations rapidly, and then is terminated by a short period of sexual reproduction
stimulated by adverse physiological conditions. Mictic zygotes are capable of resisting considerable
thermal and desiccation stress. This pattern is variable, however, and some rotifer species appear to only
reproduce parthenogenetically. Males are not known in these species.
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Because rotifer lifecycles contain a resting (mictic) egg stage able to withstand drastic conditions,
they are often the numerically dominant animal in temporary aquatic habitats such as puddles. When these
habitats are dry, the mictic zygotes are present and mature to amictic female rotifers under an appropriate
stimulus. This change can happen within an hour and is generally in response to moisture and/or food.
Today we will try to find rotifers in dried puddles if it hasn't rained lately, or we will try to find them in an
active state in puddles.
Procedure: Try to think of an ideal location nearby where temporary bodies of water are common.
Flat rooftops generally work well. If you know a good location, go there and procure a small amount of
sediment or detritus in a 250 ml beaker. On returning to the laboratory, divide the sediment equally
between two 250 ml beakers. Add approximately 100 ml of distilled water to the beakers, and seed one of
the cultures with a couple drops of Chlamydomonas culture and a dash of yeast. Start this experiment at
the beginning of the laboratory period and examine drops of the culture under the compound microscope
at the end of the laboratory. Whether or not you find anything at the end of the lab period, label your
beakers clearly and we will incubate them at room temperature for a week and examine them during next
week's laboratory.
PHYLUM ACANTHOCEPHALA
Members of the phylum Acanthocephala are commonly known as “spiny-headed worms.” There are about
1,100 species known. You probably wouldn’t guess it from looking at them, but studies have indicated
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that acanthocephalans are so genetically-similar to rotifers that perhaps they should properly be classified
as highly modified members of the phylum Rotifera. (Some authors call acanthocephalans “evil rotifers.”)
Most acanthocephalans are only a few millimeters long, but a few species can be more than a
meter long at maturity. All known acanthocephalans are endoparasites. Adults live in the intestines of
vertebrates, especially fishes. Larval acanthocephalans parasitize arthropods – either insects or
crustaceans, depending on the species.
The most distinctive feature of an adult acanthocephalan is the cylindrical proboscis that projects
from the worm’s anterior end. The proboscis is invaginable – that is, it can be inverted and withdrawn
into the proboscis receptacle using the retractor muscles – but its most noticeable characteristic is that
it’s covered in recurved spines. These spines allow adult acanthocephalans to attach themselves to the
intestinal linings of their hosts. Two sac-like projections called the lemnisci run into the cavity of the
body, alongside the proboscis cavity. Each consists of a prolongation of the syncytial material of the
proboscis skin, penetrated by canals and sheathed with a muscular coat. They act as reservoirs into which
the fluid which is used to keep the proboscis "erect" can withdraw when it is retracted, and from which
the fluid can be driven out when it is wished to expand the proboscis.
Like its rotifer cousins, an acanthocephalan has a syncytial epidermis, properly known as the
tegument. Also like rotifers, acanthocephalans have no respiratory or true circulatory systems. Gas
exchange occurs across the tegument. Smaller acanthocephalans lack any sort of excretory system, but
larger species possess protonephridia with flame cells, just as do many rotifers.
While there is no heart and no true circulatory system, a series of fluid-filled canals called a
lacunar system extends through the tegument and even into body muscles. Contraction of body muscles
helps to move fluid through these canals, and the fluid may serve to transport nutrients, gases and
metabolic wastes throughout the body in somewhat the same way that a true circulatory system does.
An acanthocephalan has no digestive tract. Since it lives inside the digestive tract of its host, it
can absorb the nutrients it requires across its tegument, in much the same way that a cestode flatworm
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does.
A spiny-headed worm has an extremely simple nervous system and very few sense organs. You
would probably expect this, given that the worm spends its entire life inside the bodies of other animals –
it isn’t as if it has to hunt for food or worry about evading predators.
Like many other endoparasites, some acanthocephalans can apparently manipulate their hosts’
behaviors. In particular, many acanthocephalan species appear to manipulate the behavior of their
intermediate hosts in such a way as to increase their chances of getting into their definitive hosts.
PHYLUM NEMATODA
Nematodes are an extremely ubiquitous group of organisms which are found in almost every conceivable
habitat. They outnumber all other groups of species except for the insects which they probably surpass in
total biomass. The majority of nematodes are less than 2.5 mm in length and are often microscopic. In the
laboratory we will examine Ascaris, which is an intestinal parasite of domesticated animals and humans.
Ascaris is large and nicely illustrates nematode body structure, which is very uniform among species.
Since Ascaris is a parasite of humans, care must be taken in handling it in the laboratory. Even when fixed
in formalin, the eggs of a female Ascaris may remain viable for a number of years.
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Nematodes are generally cylindrical and tapered at both ends and when alive, move actively and
continuously. Obtain a preserved male and female Ascaris and examine the external appearance. Note that
the body is covered by a thin, transparent acellular layer. This is the cuticle, which is secreted by a
syncytial layer of cells that underlies it called the hypodermic. The body is unsegmented, although in
some nematodes the cuticle may be ridged. The sexes are separate in all nematodes, and there is generally
a sexual dimorphism in size, with females larger than males. Can you suggest a reason for this sexual
dimorphism, Ascaris males are easily recognized by their small size and curved posterior end with
copulatory spicules. Can you determine a difference between the dorsal and ventral surfaces of the worm?
Note the lateral line and the tripartite lips
Procedure: Pin an Ascaris specimen, dorsal side up, on a wax-bottom dissecting pan. Cover the worm
with a thin layer of water or continually moisten the internal organs once you cut it open. Make a straight
dorsal cut in the worm extending from the anterior to posterior end and pin the detached cuticle to the pan.
(Be careful not to damage the internal organs when making your incision).
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First, examine the digestive system. Carefully tease out the reproductive system to expose the
intestine. Trace out the alimentary canal: mouth, esophagus, pharynx, intestine, and anus. Note the
relatively straight-tube construction of the gut that is nonmuscular except for the pharynx. The
esophagus is highly glandular. In the living Ascaris the gut is under high pressure due to the coelomic
fluid and the pharynx is responsible for pumping food against this pressure through the system.
The muscular system consists of large, elongate contractile cells with longitudinal fibers which
form specialized units on the dorsal and ventral body walls. Projections from these cells enter the nerve
cords enclosed in the lateral lines. Locate some of these muscle cells under the cuticle and examine them
microscopically (slide). Careful dissection around the pharynx may reveal the nerve ring
(circumpharyngeal ganglion) lying at an angle around the gut about halfway between the head and the end
of the pharynx.
The excretory system consists of paired excretory tubes, one passing along each lateral line. Their
function, as in flatworms, appears to be chiefly osmoregulation. The excretory tubules connect to a
specialized pair of extremely large excretory cells in the pharyngeal region. Collected fluids drain out a
nearby excretory pore. The pore may not be visible in your dissection, but you should be able to locate the
excretory tubes and possibly the excretory cells as well.
Examine the arrangement of the internal organs in a prepared cross section under the microscope.
How do the organs lie free in the body cavity?
Nemotodes, like other aschelminthes, are covered by a nonliving, acellular protective covering
called a cuticle. The cuticle of nematodes is a complex four-layered structure. The main component is
inelastic collagen giving the cuticle the properties of impermeability and rigid strength. For movement to
occur, however, the cuticle must be elastic, and the collagen is laid down as a spiral latticework of
parallelograms with protein in between. Movement of the cuticle results ln a change in the angle between
the fibers of the lattice. Peel a piece of cuticle from your dissected Ascaris. Make a wet mount and
examine it under the microscope. Can you see the plates and layers? Since cuticles form relatively rigid
exoskeleton, they must be periodically shed (molted) to allow for growth.
Nematodes do not have a circular muscle layer. Movement is brought about by contraction of
longitudinal muscles only which are opposed by pressure within the pseudocoel. In life the pseudocoel is
filled with fluid under pressure. A small puncture in the body of a living Ascaris will produce a sharp jet
of pseudocoelomic fluid, demonstrating the high internal turgor pressure under which the fluid is held.
The fluid-filled muscular tube thus forms a highly developed hydrostatic skeleton, which maintains body
shape and is necessary for locomotion. Unlike ordinary striated muscles, which act between two points of
attachment, the muscles of Ascaris contract in groups, called fields, which produce local shortening of the
body wall. Because the volume on internal fluid is constant and incompressible, the internal pressure in
the pseudocoel is increased, which causes extension of the muscle cells in other regions of the body.
Through this system the dorsal and ventral muscles act as antagonists, producing sinusoidal waves along
the length of the body, propelling the organism. For this method of locomotion to work the external
medium must be rather viscous (especially for larger nematodes such as Ascaris). Examine the
locomotion of Turbatrix in different mediums. Try distilled water, seawater, and seawater thickened with
5X methyl cellulose. Does the pattern of body movements change? Does the locomotion rate or direction
change? Can you quantify any differences?
Now return to your dissected Ascaris to examine the reproductive system. Nematodes are
dioecious (sexes are separate), and you should be sure to get a good look at both male and female
anatomy.
The male reproductive system is a single, long, highly differentiated tube. The thin terminal
portion (blind sac) is the testis where sperm are produced. Sperm move from the testis into a sperm duct
(vas deferens) that enlarges to form the larger seminal vesicle. During copulation, sperm stored in the
seminal vesicle are discharged via the ejaculatory duct out the cloaca (combined excretory reproductive
opening. A pair of spicules that protrude from the male pore functionally opens the female aperture during
copulation. Try to locate sperm cells in the seminal vesicle. The sperm of nematodes are not flagellated,
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but move by amoeboid action. Amoeboid sperm are thought to represent a special adaptation of
nematodes. What is the functional significance of nematode amoeboid sperm?
The female reproductive system of Ascaris is far larger than that of males. Notice, however, that
the basic organization of the two systems is essentially the same: a greatly modified continuous tube
arrangement. Trace the female system from its external opening, the genital pore, to the vagina, a short
heavy tube formed by the fusion of the two large uteri. Each uterus connects to an oviduct, each of which
ends in a greatly elongated, fine tube, the ovary. The ovaries form a mass of coils about the rest of the
reproductive system and intestine, to form a large proportion or the worm’s interior bulk. It may be
diffcult to visualize the entire system as simply two long tubes joined at the vagina. Tease one tube out to
try to demonstrate this.
Eggs formed in the end of each ovary are forced by pressure from masses of younger eggs behind them to
move down the reproductive tube into oviducts and uteri. Near the point where the oviduct bends back on
itself, and enlarges to form the egg-storing uterus, there is a swollen portion, the seminal receptacle.
During a female's lifetime, millions of eggs form and start their migration down the tube, passing through
the seminal receptacle. There they are fertilized by sperm stored from a previous copulation. The
fertilized eggs even undergo meiotic division and the male and female pronuclei fuse. The newly
fertilized eggs are then coated to form mature, heavy-shelled eggs that fill the uteri and are periodically
forced through the vagina and out the genital pore. These eggs become enmeshed in the intestinal contents
of the host and are passed in feces to the soil where development continues. Within the highly resistant
eggshells, the larvae develop, grow, and mole to form the next larval stage. Strictly speaking, the egg is
now no longer an egg, but an encased infective larva. Infection of the vertebrate host occurs after
ingestion.
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PHYLUM ECTOPROCTA
This picture shows the locations of mouth, anus, lophophore, funiculus and many other features typical in
a bryozoan. A lophophore is the ciliated tentacles at the top. They are food gathering structures that can be
flower like (the image) or collapsed. The funiculus is a tendon which joins the end of the gut to the colony
wall. It is the site of statoblast production and spermatogenesis. The inconspicuously located Central
Nerve Ganglion has a major nerve tract extending into each arm of the lophophore. An exocyst is the
chitin made outer layer of the body wall. And the endocyst is the inner layer.
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