Chapter 7: Phylum Annelida
Class Oligochaeta
Family: Lumbricidae
Species: Lumbricus terrestris
The earthworm, or nightcrawler, presents a typical body plan with bilateral
symmetry, closed circulatory system, metamerism, complete digestive system, and
almost all of the functional units well-illustrated by organ systems. It is the intent of
this laboratory to study the earthworm in great detail. This will mean studying
macroscopic and microscopic morphology through all body regions and physiology
of certain functional units.
The morphology of the earthworm is illustrated throughout this chapter.
Refer to these diagrams throughout your study. Living specimens are preferable. It
is necessary that you first become acquainted with the gross morphology of the
earthworm before you endeavor to correlate it with the microanatomy in the next
exercise.
Gross Anatomy
External Anatomy
One of the hallmark characteristics of Phylum Annelida is the presence of
metamerism, or a division of the body into repeated similar segments. In the
earthworm, metamerism is exemplified in several ways. First, it is obvious that
Lumbricus displays a visible external segmentation. Internally, the repetition of
certain structures (nephridia, septa, ganglia, etc.) also is evidence of metamerism.
The intersegmental furrow between adjacent external segments connects internally
to a thin, muscular septum that separates each metamere.
This organism, as in other Annelids, has a complete digestive system,
meaning it has both a mouth and an anus. These are both terminal with the ends
distinguishable by the proximity of the clitellum, a reproductive structure to the
anterior end. Also, the mouth, which is at the anterior end, has a lip-like structure
called a prostomium. It looks like half a segment. The prostomium is dorsal to the
mouth. The prostomium is not regarded as a true segment and is not counted as
such; thus, it is considered segment zero. Segment 1 directly encloses and encircles
the mouth; it is also called the peristomium. If you observe the clitellum, it is
evident that it does not completely encircle the organism as its ventral surface is
incomplete. Almost every segment in Lumbricus has eight setae. These are located
ventrolaterally, and can be felt by running the fingers down that region of the
earthworm. Each seta is enclosed in a setal sac, or chaetigerous sac, and is moved
by striated muscles that extend (protractor muscles) or withdraw (retractor
muscles) the rigid structures.
46 In addition to the mouth and anus, there are several other orifices in the
earthworm body wall. The most prominent of these are the paired male genital
pores on the ventral side of the fifteenth segment. On the fourteenth segment and in
line with the male openings are a pair female genital pores, which are smaller and
more difficult to see. Also, on the ventral surface of the earthworm and anterior to
the genital pores, are the four openings of the seminal receptacles, which are found
in the intersegmental furrows between segments 9 and 10 and segments 10 and 11.
Each segment also has an unpaired dorsal pore in the mid-dorsal line. These are
difficult to see, but will be seen later in the cross-sections. They connect the coelom,
or internal body cavity, with the exterior, and exude coelomic fluid when the
earthworm is sufficiently irritated.
Internal Anatomy
Figure 7.1. Internal anatomy of the earthworm, as you will see in dissection. Use as
a guide while completing your dissection.
You will be given a living active earthworm killed in 70% ethanol. Move the
earthworm to locate the setae on the ventral surface; this surface is also slightly
flattened. Move the worm to a dissecting pan. You will need a single-edged
razorblade or scalpel, forceps, two straight dissecting needles, and about 40 straight
pins or dissecting pins. After identifying as many aspects of the external anatomy as
possible, begin a dissection of the earthworm as follows:
1. Place the worm with its ventral side down.
2. Put a pin through the middle of the worm at segment one from the anterior end.
Stretch the worm posteriorly from this pin and put a second pin through the
body about ten segments posterior to the clitellum. Both pins should be
continued into the paraffin of the pan so the worm is held firmly in place.
47 3. With a new single-edged razorblade or scalpel, begin cutting, slightly to one
side of median, a shallow dorsal longitudinal incision from posterior to the
first pin to about four segments anterior to the posterior pin.
4. Reflect and pin down to the paraffin one side of the body wall. There will be
membranous walls called septa between each segment making it difficult for you to
carry out the operation. These can be removed by passing a pin or needle through
them close to the body wall.
5. Reflect the opposite side of the body wall; again, it will be necessary to tear septa
with a pin. If the septa are not torn, they will depress the organs and present an
atypical, flattened orientation.
6. Remove the anterior medial pin and continue the cutting and reflecting to the
anterior end. Make certain your cut is only through the body wall.
7. Along one side of the earthworm, place pins at the end of the fifth, tenth,
fifteenth, and twentieth segment. These will be used for points of reference and
eliminate the need of counting segments each time you are looking for an
organ.
8. Along the dorsal midline of the gut, there is a dorsal blood vessel (Fig. 7.1). This
vessel is responsible for most of the force needed to transport the blood through the
circulatory system. Follow this vessel anteriorly to the esophageal region. In this
region, there are five pairs of curved blood vessels called aortic arches, which
encircle the esophagus and converge ventrally to form the ventral blood vessel.
Look for the five pairs of aortic arches.
9. Dorsally in the third segment is a white, bilobed structure that is actually two
connected structures, the suprapharyngeal ganglia of the earthworm. It rests
atop the pharynx, immediately dorsal to it. These paired cephalic ganglia are
located in the head region; however, they do not comprise a true brain. Thus,
the term ganglion is used to designate a collection of neuronal cell bodies or
somata that are outside the central nervous system. We will see that most
invertebrates lack true brains and will have various ganglia in their place.
Connecting to each suprapharyngeal ganglion is a small nerve fiber, a prostomial
nerve, which predominantly carries afferent, sensory neural impulses posteriorly
from the prostomium to the suprapharyngeal ganglion. Two large nerve trunks
course ventrally, one on each side of the pharynx, from the suprapharyngeal ganglia
to the subpharyngeal ganglion, which is ventral to the pharynx. The two nerve
trunks are called the circumpharyngeal connectives. The subpharyngeal ganglion
is connected via a ventral nerve cord to a segmental ganglion in the ventral aspect
48 of each metamere. Note that ganglia can be hard to see in a gross dissection of a
worm, but the nerve cord is a prevalent, easy-to-see, structure.
10. The most prominent structures in the body cavity, which is called the coelom
belong to the digestive system, or alimentary canal. It has the regions indicated by
the sketch in the text (Fig. 6.1) and in the segments indicated. Locate each of the
digestive organs and determine their functions from the text. Overview of the
earthworm digestive flow:
mouth
buccal cavity
pharynx
esophagus
anus
crop
gizzard
intestine
11. In the 10th to 12th segment, there are lateral bulges in the esophagus. These are
the calciferous glands, and contain great amounts of calcium. They are orange or
white in living specimens and are built into the wall of the esophagus.
12. The largest reproductive structures recognizable are the large, white, trilobed
seminal vesicles.
13. Make a transverse incision through the intestine three segments anterior to the
posterior medial pin. Do not cut through the ventral blood vessel found
immediately under the intestine. Hold the intestine with a forceps and lift it as you
cut, or carefully tear, its connections with the septa. The ventral vessel should
remain intact. Continue this procedure anteriorly and observe the ventral
connection of each of the five aortic arches. As you cut, leave the reproductive
organs attached to the body wall. The procedure should continue to the brain
region where the circumpharyngeal connectives will meet to form the ventral,
subpharyngeal ganglion.
14. Ventral to the ventral blood vessel is a white, ganglionated ventral nerve cord.
The most anteriorly located ganglion in this cord is the previously-mentioned
subpharyngeal ganglion. Find this ganglion and follow the circumpharyngeal
connectives up to the suprapharyngeal ganglia,
the primitive “brain.” Note again that these structures may be hard to see, but will
be visible in longitudinal microscope slides.
15. In each metamere, coming out from the nerve cord, there are three pairs of
nerves.
16. In each segment between the septa is a pair of nephridia, or kidneys. They are
visible without magnification and are larger in some segments than in others
Each nephridium removes wastes from the segment immediately anterior to it. This
is done by means of a nephrostome projecting through the septum. The funnel
leads through the septum into the next posterior segment. The nephridium in this
49 segment has two lobes containing small tubules, which course in and out of each
lobe. Eventually, the tubule emerges and opens into a bladder then to a
nephridiopore to the outside of the earthworm. The nephridiopore is located just
posterior to the septum that the nephrostome goes through. Therefore, each segment
has another pair of orifices not previously mentioned. Locate the nephrostome,
nephridial tubules and bladder. Note that these structures are very hard to see in
a dissected state. Overview of the earthworm excretory flow:
nephrostome
nephridial tubules
bladder
nephridopore
17. You have already looked at, or noted, the seminal vesicles. These are large
structures in which dorsally sperm is maturing and ventrally sperm is stored
and matures. With the gut removed, you should see their connection to the
body wall. Protozoan parasites (clear lemon- shaped Monocystis lumbrici) are
always present.
18. The testis and sperm funnel are located ventrally in the seminal vesicles. To
see these you will have to flush out the seminal vesicles well. Sperm is conveyed to
the outside through the sperm funnel, vas efferens, and vas deferens to the male
pore. Overview of the earthworm male reproductive flow:
testes
seminal vesicles
sperm funnel vas efferens
genital pore
vas deferens
male
19. During copulation, sperm is transferred from the male pore of one earthworm to
the seminal receptacles of another. The seminal receptacles are two pairs of white,
small ventral structures located in segments 9 and 10. Sperm are stored in these
reservoirs until voided to fertilize eggs.
seminal receptacle
seminal receptacle openings
20. Segment 13 contains the remainder of the female reproductive system.
In the ventral body wall, just lateral to the nerve cord, are two small ovaries
attached to the anterior septum. At the same location but attached to the
posterior septum are the two ciliated funnels, which empty into the oviducts,
which go through the septum and open through the female pore in the 14th
segment. It is doubtful that you can find these because they are so small, even
microscopically. It is a class challenge. Please alert one of the lab instructors if you
think that you have found the female genital pores. Overview of the earthworm
female reproductive flow:
ovaries
coelom
ciliated (oviducal) funnel oviduct
⇅ ovasac (stores eggs)
50 female genital pore
21. At the end of copulation (after the transfer of sperm) the earthworms separate,
each containing sperm from the mate in their seminal receptacles. The
clitellum then produces a collar, which peristaltic movements of the earthworm
cause to move forward as a sheath closely applied to the cuticle of the earthworm.
When the level of the fourteenth segment is reached, the eggs are deposited in this
sheath. The collar continues to move anteriorly. Sperm are added to the eggs in the
collar in the 9th and 10th segments. The collar continues forward, finally moving
completely off the anterior end of the worm. The ends of the collar constrict as they
leave the worm, and the collar then hardens into a cocoon, from in which the young
worms develop and eventually emerge. Earthworm cocoons are on demonstration.
22. Look at a longitudinal section of an earthworm through a dissecting
microscope and identify digestive, nervous, reproductive, and circulatory
structures. This general view will introduce you to the histology of the
earthworm which will be covered later in this laboratory session.
Laboratory Approach
1. Read exercise before coming to laboratory.
2. Listen and take notes on oral laboratory introduction.
3. Spend a few minutes organizing your approach to the exercise in your mind.
4. Make neat, organized notes, drawings, and references on back of
appropriate pages in the laboratory directions.
5. Correlate the elements (lecture, laboratory, references) into an integrated
cohesive picture. 51 Chapter 8: Microanatomy of Lumbricus terrestris Introduction There are a great variety of slides from different regions of the earthworms, so it will take some effort to find all structures. Follow these directions in order. 1. Identify all the tissues and organs in the reference cross-­‐section through the intestine. 2. Go back over the structures in the reference cross-­‐section and investigate the cellular detail where requested. 3. Compare the other sections with the reference cross-­‐section. 4. Review and reinforce as if you were to be examined on the material at the end of the class period. Histology Figure 8.1. Cross-­‐section through the intestinal region (“Posterior to clitellum”). Use as a guide to reference the terms below. The following structures should be recognized in the reference cross-­‐
sections through the intestine (Fig 8.1): • Cuticle: non-­‐cellular layer secreted by epidermis; 80% protein—stains homogeneously • Epidermis: outer single layer of cells. Cells of four types: o Albuminous cells: stain light in color, extend from muscle to outside o Mucous cells: darker staining, extend from muscle to cuticle o Supporting cells: rectangular cells, nuclei half distance to cuticle 52 •
•
•
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•
•
•
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o Basal cells: small, triangular cells resting on outer muscle layer of body wall; cells do not extend to cuticle Muscle layers: demonstrates antagonistic actions of muscles o Circular muscle fibers: outer and thin; contraction decreases diameter of body and lengthens earthworm o Longitudinal muscle fibers: inner and thick; fibers look like feathers when cut in cross-­‐section, contraction shortens body Peritoneum: one or two cells thick; an epithelium that lines body cavity covers nephridia, blood vessels, nerve cord, body wall, internal organs, etc.; composed of two contiguous parts: o Visceral peritoneum: covers central internal organs like digestive tract; comprises most central portion of eucoelom o Parietal peritoneum: lines inner surface of body wall; comprises most peripheral portion of eucoelom Setae: pairs of setae interrupt body wall in four places; interruptions in muscle usually seen but perhaps one seta of the eight in the segment is visible Intestine: fills center of section; note that its cavity (lumen) is frequently odd-­‐shaped due to the collapse of the wall of the intestine during histological preparation of the tissue Typhlosole: invagination in dorsal intestinal wall to increase surface area Chloragogue cells: on coelom side of intestinal wall; comprise the visceral peritoneum in intestinal region; cells stain palely because, while the specimen is alive, they contain lipids which are removed during histological preparation of the tissue; probable source of coelomocyte Muscle layer medial to chloragogue cells: this layer is smooth muscle. Metanephridia: also known as kidneys, loosely oriented tissue in coelom. Sometimes a nephridiopore is present. Nerve cord: three giant nerve fibers in dorsal part of nerve cord. Cell bodies of many neurons in ventral part of cord. Included in the covering of the nerve cord are two lateroneural blood vessels and one subneural blood vessel. Coelom: formed by splitting of solid mass of mesoderm Comparison with other cross-­‐sections After obtaining an understanding of the cross-­‐section through the intestinal region look at the cross-­‐sections mentioned, compare the following with the intestinal region. 53 Figure 8.3. Cross-­‐section through the pharyngeal region. • Nephridia not present • Very thick muscular wall • Epithelial cells that line pharynx look bubbly • Suprapharyngeal and subpharyngeal ganglia present; anterior to nerve cord Figure 8.4. Cross-­‐section through the aortic arches, or esophageal region. • Aortic arches surround the esophagus 54 Figure 8.5. Cross-­‐section through the esophagus. • Recognize calciferous glands with their distinctive appearance. Septa within glands have appearance of "barbed wire" Figure 8.6. Cross-­‐section through the crop. 55 Figure 8.7. Cross-­‐section through the female reproductive organs, found in the esophageal region. • Ovaries: represented by two small basophilic ventral structures that contain growing follicles: • Oviduct Figure 8.8. Cross-­‐section through the male reproductive organs, also found in the esophageal region. 56 • Seminal vesicles-­‐-­‐large bodies with heterogeneous, purple staining. Dark staining cells are sperm in different stages of development. Light staining vesicles containing very darkly-­‐staining, small masses are protozoan symbionts, Monocystis lumbrici. • Testis: enclosed in and ventral to seminal vesicle. Compact, stains darker and more homogeneously than seminal vesicle. Frequently looks like a bow tie. Has narrow portion ventral to nerve cord. • Sperm funnel: has a labyrinthine nature. Very dark staining due to numerous sperm nuclei being aligned. Figure 8.9. Vas deferens and vas efferens. • Vas efferens: sperm funnel tapers to form vas efferens in the coelom. Often see multiple round or oval sections with long cilia in lumen. Vas efferens do not come into contact with body wall. • Vas deferens: formed by union of two vasa efferentia on each side of the midline. Soon after vas deferens is formed, it is applied directly to inner surface of body wall. As approach male pore, vas deferens will sink into body wall musculature. Figure 8.10. Cross-­‐section through the clitellum. • Recall this is the thick collar around the earthworm. • The outer epithelial is largely responsible for the thickness 57 Figure 8.11. Cross-­‐section through setae. Longitudinal Sections of Lumbricus Again, look at a slide of the earthworm in longitudinal section and identify as many parts that we have studied as you can. Longitudinal sections are helpful because they show features that appear on many different cross-­‐
sections. Make as many sketches as you like. Use the projection microscope to help trace typical longitudinal sections. Review of the Earthworm A good correlative approach to the gross and microanatomy is to picture structurally and functionally how organs are related to one another. A satisfactory device to use for this purpose is to trace various pathways in an organism. Refer to Chapter 7 for pathway flows for different organ systems. Materials 1. Preserved specimens of class representatives. 2. Prepared slides of cross-­‐sections (intestine, mouth, pharynx, aortic arch region, gizzard region, crop region, ovary region, male sex organs, calciferous glands, dorsal pore, nephridiopore, clitellar region). 3. Prepared slides of sagittal sections of Lumbricus. 4. Slides of Nereis parapodia, w.m. 5. Slides of Leech, w.m. and x.s. 58 Phylum Review Phylum Annelida • body in segments (metameres or somites) • chitinous setae • schizocoelomate form of eucoelomate • complete digestive system • closed circulatory system • hermaphroditic (monoecious) Class Oligochaeta (means "few long hairs") o Lumbricus terrestris o no terminal suckers o setae few per segment (usually 8) o no parapodia o clitellum (only in sexually mature individuals) o terrestrial, freshwater habitats Class Hirudinea (means "leech") o Hirudo medicinalis o leeches o terminal suckers o no setae o no parapodia o clitellum (only in sexually mature individuals) o terrestrial, freshwater, marine habitats Figure 8.13. Whole mount of a leech. Class Polychaeta (means "many long hairs") o Nereis veirens (clam worm) o no terminal sucker o most segments with parapodia bearing tufts of setae o mostly marine o cephalization-­‐-­‐distinct head bearing eyes and tentacles 59 Figure 8.13. Parapodia of a polychete, Nereis. 60