by  Peggy Brown (Newburg High School, Newburg, MO) and   Madeline Niccore (Longmont High School, Longmont, CO)

 

• Students perform an experiment to compare the sensitivity of the various developmental stages of Red Flour Beetles (Tribolium castaneum) to ultraviolet (UV) radiation.  Larvae, pupae and adult beetles are exposed to a source of UV-B radiation for 0, 15, and 30 minutes per day for a week.  They compare the mortality of the various stages at these time periods.  Students should be able to predict that the larvae and pupae will be more sensitive than the adults and account for the differences.

• Students make a hypothesis, conduct an experiment and analyze data comparing the effects of UV-B radiation on the various developmental stages of Red Flour Beetles.
• This investigation can be adapted to various levels of biology.  For lower level classes fairly specific directions for setting up the laboratory can be given.  An example of this type of investigation is provided.  More experienced students might design their own procedures.
• Students will work in groups of about 4 on this experiment.
• There are numerous extensions of this experiment for group or individual research projects.

• Biology students

• Grade Level 9 - 12

Notes to the Teacher: to top

1. Required of students - Understanding of the stages present in complete metamorphosis.
2. Preparation time needed - After initial acquisition of UV-B radiation source and sufficient classroom supply of Red flour Beetles, there in minimal preparation time.  Obtain beetles at least several weeks before performing the experiment.   About 30 minutes would be required for gathering materials for lab.
3. Class time needed - One 50 minute class period to separate beetles and set up experiment. Additional 5 - 10 minutes per day for a total of 5 days to observe and prepare beetles for exposure.  One additional 30 minute time period to make final observations and work on lab report.
4. Hazards/Precautions - Fluorescent UV-B lamps should be inside a UV Irradiation Chamber.  If using a Quartz-Halogen Security Lamp, students should be warned not to look directly at the light and be required to wear UV protective goggles. Also, Quartz-Halogen Security Lamps give off a great deal of heat.  Care must be taken that no combustible material will have contact with the lamp when it is on.

Red Flour Beetles

    Description
 
    Red Flour Beetles (Tribolium castaneum) are a common pest in flour and other grains.  Red flour beetles show complete metamorphosis changing from egg to larva to pupa to adult. Newly emerged adults require several days to complete the processes of hardening and darkening of the exoskelton and to reach sexual maturation.  The duration of each stage of development time is temperature-dependent.  The table below lists duration of developmental stages at various temperatures.  The beetles grow at room temperature although somewhat slower.

            Developmental Intervals (days) of Red Flour Beetles
           _________________________________________

                                    25^o          30^o           34^o
            egg                     5              4              3
            larva                32            18             13
            pupa                  8              6               5
            Total                45            28            21

    Care and Handling

    Red Flour Beetles can be easily maintained in the classroom and require no special conditions.  They can be grown in a glass jar containing white flour enriched with a small amount of brewer's yeast.  Red flour beetles can be obtained from various supply houses or contact Peggy Brown. The larvae, pupa and adults are easily separated from the flour with a tea strainer. The collected beetles can be then transferred into a petri dish and the developmental stages identified and separated.  Groups of beetles can be transferred from one dish to another using a soft bristle paint brush.  Individual organisms can be transferred using a "bug sucker" or individual bug handler. This devise is made using a piece of 1/8 inch latex tubing with one end stretched over a disposable small pipet tip.  The pipet tip has to be cut off at a point where its diameter is slightly greater than the tubing and then inserted into one end.   At the other end, place a large pipet tip.  A disposable straw can fit into the large tip so that it can be used by different students.  A small amount of suction at the end of the straw will pick up and transfer individual beetles.  This brief summary of handling beetles was adapted from information provided by Dr. Richard Beeman of the USDA, U.S. Grain Marketing Research Laboratory in Manhattan, KS.   Additional information about care and handling and pictures of Red Flour Beetles can be obtained on the Tribolium Home Page at
 http://bru.usgmrl.ksu.edu/beeman/tribolium.html.

Ultraviolet Radiation

    Ultraviolet (UV) radiation can be absorbed by molecules in the cell producing specific chemical changes.  The most common chemical change produced when DNA absorbs UV is the production of a covalent bond between adjacent pyrimidines resulting in a pyrimidine dimer.  These dimers disrupt the replication of DNA.  DNA repair pathways are present in all organisms including red flour beetles and humans.  One repair pathway called error-prone repair can sometimes lead to changes in the DNA sequence producing mutations.
    UV radiation is divided into 3 ranges known as UV A (400 - 320 nm), UV-B (320 - 290 nm) and UV C (290 - 180 nm).  Peak absorption for DNA is in the UV-C range which is emitted by germicidal lights.  UV-C emitted by the sun is strongly absorbed by the ozone layer.  The DNA absorption spectrum extends down into the UV-B range.  UV-B emitted by the sun is only partially absorbed by the ozone layer.  This is the type of UV radiation most associated with the development of skin cancer in humans.  UV-A which reaches the surface of the earth is not absorbed by DNA.  For more information on UV radiation, DNA repair and the interaction of UV with the Ozone layer see http://www.phys.ksu.edu/gene/chapters.html.

Classroom Sources of  UV Radiation

    Various sources of UV-B radiation are available for classroom use.  Fluorescent lamps that emit UV-B are commonly used for observing fluorescent dyes in DNA and may already be available in your classroom. Fluorescent UV-B lamps  are available from a number of suppliers such as Cole Parmer Instrument Company (800-323-4340).  A 15 Watt UV-B (312 nm) tube is under the catalogue number G09815-63 and sells for about $30.  A fluorescent UV-B lamp should be mounted inside a protective enclosure. Directions for building a UV Irradiation Chamber are available at http://www.phy.ksu.edu/gene/RAD.html.
    A less intense source of UV-B is a 300 Watt Quartz-halogen lamp used as an outdoor security light and sold at hardware or discount stores for about $10.  These emit a considerable amount of UV-A, UV-B, and even some UV-C.  The light must be wired to a power cord and mounted on a stand at least 8 inches above a petri dish.  Suggestions for mounting a security lamp are given on the web at http://raven.umnh.utah.edu/new/teachkits/disease/light.dir.html.
    Normally a protective glass absorbs the damaging UV photons.   Removal of this protective glass cover becomes a source of UV radiation similar to the spectrum present in sunlight.  Since the lamp produces a considerable amount of heat, the lamp must be at least 8 inches above the petri dish.  The lamp with the glass cover on would provide a suitable control.  Students should be warned not to look directly at the lamp and should wear UV blocking safety goggles. Caution should be taken that combustible material is not allowed to touch the lamp when it is on.

Sources of Other Types of UV Radiation

    Students may want to use other sources of UV radiation as extensions to the lab.  A germicidal lamp provides a source of UV-C.  These lamps may be present in cabinets to sterilize goggles.  Germicidal lamps are available from suppliers such as Cole Parmer.  These lamps give off high energy UV radiation and must be mounted in a UV Irradiation Chamber.  Time of exposure would need to be adjusted because of the higher energy content of  UV-C.
    UV-A sources sold as "black lights" probably would not affect the mortality and development of the beetles since it is not absorbed by DNA..

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Introduction:  The Earth's surface is receiving increased amounts of Ultraviolet (UV)-B (290 - 320 nm)  due to depletion of the ozone layer.  UV-B can be absorbed by DNA and produces specific chemical changes which disrupt the structure of the DNA.  This can cause death to the cell or lead to mutations (changes in DNA sequences).    In humans exposure to UV-B radiation is associated with increased skin cancer rates.  Other organisms can be adversely affected by exposure to UV-B.  They may be more sensitive to damage from the radiation at different times in their life cycle due to changes in growth rates and differences in protective coverings or pigmentation.
    Red Flour Beetles (Tribolium castaneum) show complete metamorphosis going from egg to larva to pupa to adult.  In this experiment the various developmental stages of these beetles will be exposed to a source of UV-B Radiation for different time periods for a week in order to compare the sensitivity of these stages to the radiation.

Purpose: Compare the sensitivity of the developmental stages of Red Flour Beetles exposed to varying amounts of UV radiation.

Pre Lab Questions:  Do you expect there to be differences in sensitivity to UV radiation of the larvae, pupae and adults of red flour beetles and explain your answer.
 
 
 

State a hypothesis to be tested in this experiment.
 
 

Equipment: Culture of Red Flour Beetles, UV-B radiation source, small, soft bristle paint bristle, individual bug handlers, 3 small jars such as baby food jars and 4 petri dishes.

Procedure:

Caution: UV-B radiation is hazardous.  In order to avoid damage to your eye, you should NEVER  look directly at any kind of UV radiation .  If using a fluorescent UV-B lamp, it should be inside an irradiation chamber.  If using a security light as a UV-B source, do not look directly at the light and wear UV protective goggles.   Do not allow any combustible material to touch the lamp.

1. Remove some flour containing beetles from your culture jar with a spoon and place in a tea strainer..
2. Sift the flour in the tea strainer back into the jar and spread the larva, pupa and adults into a petri dish.
3. Count out 10 larvae, 10 pupae, and 10 adults and place in a clean petri dish.  Individual organisms can be handled by gently sucking on a straw inserted into an individual beetle handler. Beetles can also be transferred from one dish to another with a soft bristle paint brush. Label this petri dish 0 minutes.  This group will receive no exposure and will serve as a control.
4. Repeat step 3 and transfer the 10 of each stage into another petri dish and label 15 minutes.  Repeat step 3 again and transfer to a 3rd petri dish and label 30 minutes.
5. Place the dishes labeled 15, and 30 minutes under a UV source as directed by your teacher for the appropriate time.  Remove the cover during the time of exposure since plastic or glass may block the radiation.
6. At the end of the maximum exposure time, the beetles should be brushed into separate small glass jars containing an inch of flour.  Be sure the jars are labeled with the appropriate exposure time.
7. Construct a data table to record your observations.
8.  For the next 4 days at the beginning of the hour, separately pour the contents of the jar into a tea strainer and sift off the flour.  Place the beetles in a petri dish and count the number of  each stage that are dead.  Observe whether any of the larvae or pupae have changed to the next stage.  Expose each petri dish for the appropriate time and return to their glass jar.
9.  On the final day of  the experiment count the number dead at each stage.  Put the beetles in a collection jar as directed by the teacher and clean out your small jars and petri dishes
10. Prepare a graph of the percent mortality after 1 week of exposure to UV-B for the larvae, pupae, and adults in each exposure time group.  Plot time of exposure (0, 15and 30 minutes) on the X-axis and % mortality on the Y-axis.  For each exposure time group there should be a bar for each of the three developmental stages.

Conclusions:

1.  Describe differences that were observed among the larvae, pupae and adults to UV radiation.
 
 

2. Was your hypothesis supported in this experiment?  Explain
 
 

3. Provide possible explanations to account for the differences in UV sensitivity you observed among the developmental stages.
 
 

4. Describe any relationship you observed between the time of exposure and mortality.
 
 
 

5. As a result of your findings, form a new hypothesis for further research.
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Manney,  Tom et al, 1996, A Classroom Guide to Yeast Experiments, Kansas State University, Manhattan, KS  (Although this book primarily concerns using yeast in classroom experiments, there is excellent background material on UV radiation, the biological effects of UV, repair of DNA and it gives practical information about sources of UV lamps for the classroom. This book can be purchased for $30 plus $8 Shipping and Handling from the GENE Project, Dept. of Physics, 116 Cardwell Hall, Manhattan, KS 66506-2601.  It is also available on the website listed below.)
 

 http://www.phys.ksu.edu/gene/index.html (This website has a link to A Classroom Guide to Yeast Experiments as described above.  It also has a link to the Tribolium Home Page)

http://bru.usgmrl.ksu.edu/beeman/tribolium.html  (Tribolium Home Page)

http://raven.umnh.utah.edu/new/teachkits/disease/light.dir.html (Directions for making a Quartz-Halgoen Security Lamp into a UV source.