Hawley High School
A simple earthworm dissection lab can be enriched by reinforcing previously presented concepts and be used as a platform to introduce new material. Concepts could include symbiotic relationships, meiosis, and life cycle patterns of sporozoans. It is easy for students to observe many of the free living protozoans in lab first hand but the primary example for Apicomplexa (Sporozoans) is Plasmodium. To observe Plasmodium a series of prepared slides must be used which isolates a student from the biological system. Monocystis, a gregarine protozoan, which in in the same class as Plasmodium lives in the seminal vesicles of most earthworms. Students can prepare a simple smear from seminal vesicle tissue and stain it. All the developmental phases of gametogony and sporogony in Monocystis can usually be identified from one smear. Monocystis has a less complex life cycle using one host and does not exhibit the asexual life cycle that pathogenic apicomplexans such as Plasmodium have. Students can use what they observe to focus on the objectives for the lab without having to master complicated procedures.
There are several units into which this lab exercise could be incorporated during the year. Protists taxonomy, meiosis, sexual life cycles, comparison of reproductive cells, ecology focusing on symbiotic relationships, and microscope orientation/microtechnique. This lab does not have to be the focus for an entire class period but can be used as enrichment. There are many variations and objectives possible for this lab. Objectives should be tailored to the situation. This student lab design relates to the sexual reproductive cycle using Monocystis as a survey organism for the Apicomplexa phylum.
Target Audience or Age Group to top
Notes to the Teacher: to top
A pair of trophozoites (+ and -) will unite and encyst together in a process called syzygy. Each trophozoite is now a gamont. The gamonts are compartmentalized within a gametocyst so they retain their genetic individuality. The gamont begins to undergo fission and produces many haploid gametes (isogametes). Compartmentalization is lost and the gametes from each gamont strain begin to pair and syngamy occurs forming zygotes within the gametocyst. This is the only time during the life cycle that diploid cells are found.
Each zygote develops into an oocyst (sporocyst) and undergoes meiosis following with mitosis to produce eight spindle shaped sporozoites which are still enclosed within the lemon shaped oocyst (17-25 um long by 8-10 um wide). The oocysts can be released when the earthworm dies and decays completing the cycle when another earthworm ingests the oocyst. The sporozoites are released in the gut of the earthworm where they migrate to the seminal vesicles and mature into trophozoites. The oocysts can also be transferred through the seminal ducts when earthworms are fertilizing their eggs. Birds eating infected worms can also help disperse the oocysts. The oocysts are unharmed in birds' digestive tracts and are passed through to the soil. The Monocystis only has a sexual life cycle.
Crossword Puzzle using vocabulary
Answer Key to Crossword Puzzle
*diploid *haploid *gametes *meiosis *mitosis *parasite *zygote
Apicomplexa gametocyst gametogony gamont Monocystis oocyst sporogony
sporozoite syzygy syngamy trophozoite
*These terms should be the
focus if the students are not familiar with
these concepts. The other terms can be used as enrichment
to help reinforce the *terms
if the students are familiar with the
the background material provided. Pick out key events and diagram them
using key terms in a cyclic sequence using an entire piece of paper.
Number the key events starting with the trophozoite.
2. Take sharp pointed scissors and make a shallow, dorsal cut in the body wall from segments 10-15. Extend the cut through both sides of the body wall. The cream colored seminal vesicles should be evident. There are 3 pairs. Gently clip out a vesicle and place it on a clean slide.
3. Add a drop or two of methylene blue. Place a coverslip on the slide. Using a clean eraser gently squash the vesicle. Try not to use any twisting motion. Examine the slide for signs of Monocystis.
4. Sketch the stages of development as you locate them onto the diagram you made in step 1.
1. There are more than 70 species of Monocystsis. Is there any evidence that there is more than one species in your specimen? Explain.
2. Which is the only stage that is diploid? What is the importance of having a diploid stage in the life cycle?
3. What is the genetic difference between the haploid gamont and the haploid sporozoites from the same generation? What processes contributes to the differences?
4. Which stage is the most prevalent in your stained smear? Support your answer.
5. Malaria is caused by Plasmodium in the Apicomplexa phylum. It has a more complex life cycle that includes an added asexual development to the life cycle. The asexual part of the cycle (merogomy) amplifies the number of trophozoites exponentially before transformation into gamonts. Which parasite (Monocystis or Plasmodium) would you suspect causes more damage to the host? Explain.
6. What would you suspect the advantage of merogomy is for Plasmodium? You might want to review the life cycle of Plasmodium from your textbook to help you support your answer.
Extension/Reinforcement/Additional Ideas to top
Permanent mounts can be made of a smear so that data collected can be preserved, compared and analyzed later. Differential staining can be done in a short time using four or five small coplin staining jars.
Questions for independent student research to top
1. Is one stage of
development more common at one time of the year than another?
2. Are the different species of Monocystis host species specific?
3. What physiological changes in the worm occur when it is infected?
4. Does infection affect the reproductive fitness of the host?
Extensions beyond earthworms to top
1. Monocystis is an acephaline (aseptate) gregarine. Another type of common gregarine are the cephaline (septate) gregarinas. These are common in the intestine of grasshoppers and cockroaches. (Wallace, 1997) Comparisons in structure and life cycle could be done.
2. Students could compare and contrast the Monocystis and Plasmodium life cycles.
3. Phylogenetic relationships
could be researched using the differences of the life cycles in the Apicomplexa
phylum compared to other protozoan phyla or within the Apicomplexa
Sheridan, Philip (1986). Monocystis: An Earthworm Parasite. American Biology Teacher. 48(1).
Smyth, J.D. (1994). Introduction to Animal Parasitology. (3d ed.) Cambridge. Univ. Pr., p. 89.
Wallace, Robert L. and Walter K. Taylor (1997) Invertebrate Zoology, a Laboratory Manual. (5th ed.) Prentice-Hall, pp. 20-23.
Parasite World Wide Web Information Server
1. Smear the seminal vesicle on the slide and air dry.
2. Stain in Harris's hematoxylin for about three minutes. Time will have to be optimized for conditions.
3. Rinse briefly in tap water.
4. Add 0.1% sodium bicarbonate for about one minute or until distinctly blue in color.
5. Rinse in tap water
6. Smear may be counterstained with eosin in 70% ethyl alcohol. Once again conditions will have to be optimized. Start with 45 seconds.
7. Dip several times in 95% ethyl alcohol.
8. Air dry or dehydrate further by dipping in another 95% ethyl alcohol bath.
9. Check the quality
of the stain under a microscope. If satisfied mount in a resinous
medium such as Canadian balsam or Kleermount with a glass coverslip. Label
and allow to harden.