SEXUAL LIFE CYCLE DEVELOPMENT USING MONOCYSTIS, A PARASITIC SPOROZOAN FOUND IN EARTHWORMS

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

• Introductory Biology, Biology, Advanced Biology students
• This lesson can be tailored for all experience levels.  On a simpler level it has been used successfully with inclusion resource students in lower level biology classes. With extension exercises this lesson would be suited for advanced biology classes to help them reinforce complex concepts or as a platform to develop independent research.

Notes to the Teacher: to top

1. Required of students -  Be familiar with basic microscope techniques such as preparing a wet mount and focusing.
2. Preparation time needed -  Prep time is minimal if methylene blue stain is available as stock and dissection supplies are already organized.  Preserved earthworms can be used but I prefer large living worms from the bait store.   The day of the lab the earthworms can be prepared by rinsing

them and putting them in alcohol at least 30 minutes prior to dissection.  Inexpensive rubbing alcohol works well.
3. Class time needed - It takes 15 minutes or less to dissect the seminal vesicles of the worm out and prepare a slide. The rest of the class time available can then be used to focus on the selected objectives.
4. Hazards/Precautions -  Monocystis is host specific and is nonpathogenic to humans.   The basic precautions that you would use for a dissection lab and for handling stains should be adequate.

• preserved or living earthworms - Mature earthworms regardless of species rarely fail to contain parasites.  Living worms in late fall are the least likely to yield many parasites.  If earthworm dissection is not one of your objectives the number of worms can be reduced.  There are 3 pairs of seminal vesicles providing six lab groups with a specimen.  Seminal vesicles can even be snipped out and stored in ethanol and used later.
• sharp dissecting scissors
• methylene blue stain
• slides/coverslips
• optional - dissecting pan and pins depending on motor skills
• background information sheet
• diagram of developmental stages of Monocystis

Typical Life Cycle of Monocystis

GAMETOGONY

 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.

SPOROGONY

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.

VOCABULARY 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 concepts.
 

Classification

Kingdom:  Protista
Phylum:  Apicomplexa
Class:  Sporozoea   Subclass:  Gregarineasina
Order:  Eugregarinida
Family:  Monocystidae
Genus:  Monocystis
Species:  over 72 species most living in earthworms
 
Labeled Life Cycle Diagram
Unlabeled Diagrams of Life Cycle

INTRODUCTION

Many times there is more to something than meets the eye.  Within the seminal vesicles of earthworms live Monocystis, parasitic protozoans. These parasites belong to the phylum Apicomplexa.  Your text may still have them classified as Sporozoans. Apicomplexa have complex life cycles alternating between the production of gametes and sporozoites.  Monocystis have a relatively simple sexual life cycle with all the stages found in mature seminal vesicles.

PURPOSE
 

1.  Isolate the seminal vesicles in an earthworm and make a temporary wet mount
2. Sequence and diagram the developmental stages in the sexual life cycle of Monocystis using the background information available.
3. Locate and sketch the cells that correspond with the developmental stages in the sexual life cycle of Monocystis
4. Relate when events of meiosis and mitosis occur in the life cycle
5. Explain where in the life cycle will genetic recombination occur and why genetic recombination is important
MATERIALS
Background discussion
earthworm
sharp scissors
dissection needle
slide/coverslip
methylene blue stain
Microscope
lens paper
diagram of earthworm anatomy
diagrams of various stages in Monocystis development
T-pins (optional)
dissecting pan (optional)

PROCEDURE

1.    Read 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.
 
DISCUSSION QUESTIONS
 
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

Microtechnique extension

  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 phylum itself.

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.

Gregarine Parasite World Wide Web Information Server
 

Microtechnique

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.
 

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