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 (
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
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, t h e 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
PHYLUM ROTIFERA
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
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 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 digestive system which terminates in an anus. The digestive
system of nematodes lacks cilia. Can you suggest a reason for this difference between
nematodes and rotifers?
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.
Rotifers have unusual sexual habits. 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.
Because rotifer life cyc les 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 that acanthocephalans are so geneticallysimilar 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 body cavity – 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.
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 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.
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).
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 remove several and examine them microscopically. 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, 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.
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.