Objectives

• Determine whether the presence of neem leaves alters bean beetle (Callosobruchus maculatus) behaviors and/or has insecticidal properties.

• Determine whether a concentrated extract of neem has a similar or different effect on bean behaviors compared to neem leaves.

Introduction

Bean beetle infestation affects stored beans (Fabaceae seeds) and causes loss globally, especially in poor countries where the beans are the main source of nutrients for the population. Finding means to prevent the beetles from laying eggs on the beans while maintaining the quality of beans has been of great interest to researchers and economists. Neem, margosa, or Azadirachta indica, is a type of tree native to Southeast Asia that has a variety of uses. Parts of the tree are used as medicine, food, in hygiene practices and as an insect repellant. Your goal is to test the bioprotective properties of neem against the bean beetle by designing a series of experiments with the objectives listed above.

Materials

Adults bean beetles (Callosobruchus maculatus) will be available to you in the laboratory as will supplies of beans, neem leaves, and neem oil. An assortment of supplies for manipulating beetles and performing your experiments will be available.

Experimental Design

Does neem have a bioprotective property that deters bean beetles?

BEFORE THE LAB! These count toward your overall lab exercise grade.

• Locate two articles describing the neem tree and its potential insect repellant properties and bring these with you to the lab

Type your responses to the following and bring to the lab. Your name and the date should be on the upper right hand corner of the page.

• Describe two experimental designs for evaluating the question above and addressing the objectives of this study
• Predict the outcomes for each experiment.
• Identify and list the variables you would manipulate in each experiment.
• Identify and list the variables you would keep constant in each experiment.
• List the data you would collect to determine if your predictions were true for each experiment.
• To the best of your ability, describe the statistical analyses that you would carry out to test your predictions for each experiment.

Come to class prepared to present your experimental designs.

Literature Cited

Students will cite literature from their own searches in their final written lab exercise.

This experiment was written by Malathi Srivatsan and Amy R. Pearce, 2013 (www.beanbeetles.org).

Copyright © by Malathi Srivatsan and Amy R. Pearce, 2013. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.

For the lower-level classes, we used the first half of a 2 hour lab to discuss their experimental designs and literature. Then the entire class came up with a design (one that we 'led' them to) and then the following steps were carried out in the remainder of the first lab.

Overall Objective: To test the effectiveness of neem as a beetle deterrent and insecticide.

Week 1: Testing Bean Beetle Behavior

1. Students were placed in groups of 3-5.
2. Each group was given a set of materials and used the large petri dish which they divided into two compartments separated by a cardboard wall; the small petri dish served as a central hub (large petri dish, small petri dish, 2 pieces of cardboard, tape were used, Figure 1). Instructors may wish to recommend counterbalancing the contents of each side.
3. Using a marker they labeled one side of the lid "control" and the other side "neem". They also put a group name on the lid to identify their plate the next week.
4. Students weighed beans. (approximately 17 grams per side, but will depend on the size of the dishes).
5. They poured 17 grams of beans into the "control" side of the plate. They also counted the number of beans in this side and recorded the number underneath the "control" lid.
6. Next they weighed and poured 17 grams of beans into a Ziplock bag.
7. Then neem powder or oil was added to the bag and shaken to coat the beans. (In some of our labs, an assigned amount of neem leaf powder was used. The amount of neem was approximately either 0.5g, 0.75g, or 1.0g, but may be increased. However, when using the oil, we followed the instructions for preparation on the bottle and the beans were simply soaked or sprayed. Word of caution if using the oil, make sure you allow enough time for it to soak into the beans before exposing the beetles. If not, the beetles will get stuck in the oil.)
8. The neem/bean mixture was poured into the "neem" side of their plate. Students counted the number of beans on this side and recorded it underneath "neem" on the lid.

Figure 1. The test apparatus students used for their experiments consisted of a 150mm petri dish divided in two sections using cardboard and tape. A central 35mm dish was an untreated area for introducing beetles to the test dish from which they could move to either side of the larger dish. In this photograph, dried ground neem leaves were mixed with mung beans on the left side if this dish and the mung beans on the right side are untreated. Some ground neem leaves also are visible under the 35mm dish.

Once prepared the plates are ready for observing beetle behavior. Each group was given 3 female and 2 male beetles (could use more or less) and asked to organize themselves into as scientists into observers, timers, and recorders. Using a sorting brush, they transferred beetles one at a time into the small petri dish in the center. Beetles were allowed to acclimate to their new environment for approximately 5-10 minutes and then students recorded the time (time = 0 min). They observed how many beetles migrated to each half (control or neem) at 30-second intervals for 10 minutes and recorded their observations on the data sheet below.

Once observations were completed, they placed the covered plates with the beetles in the incubator at 28ƒ C to be used for further analyses over the next few weeks.

Other Notes:

• Students may need help grasping the concept of a positive and a negative control. For the positive control both sides were filled with 1.0 grams of neem.
• If students work in groups we recommend delegating individual responsibilities. See Neem Lab Write-Up file.
• Neem oil has a strong odor.
• If you use a brush for the neem, make sure to rinse it afterwards to prevent contamination.
• If labs meet weekly and you wish to observe oviposition, it is best to remove the adult males and females within 24-48 hours of placement.
• Discrimination tests of 24-48 hours may be sufficient to observe egg laying preferences, but permitting egg laying for longer periods may obscure choice if the preferred choice gets overly used. The presence of eggs will deter females from laying additional eggs (see the laboratory activity, Intraspecific Competition in Bean Beetles) and may induce females to lay eggs on a less preferred bean (even a toxic bean).
• In adapting this lab exercise, beetles may be tested individually or in groups. A group test is fine for direct observation of location preference, but for the reasons mentioned above, egg laying preferences would be better if conducted with females tested individually and would yield completely independent replicates.

Sample Data Sheet-adult location preference                                      Zero Time: ________

 

Time	# of Migrations to Control Side	# of Migrations to Neem Side
30 sec
60 sec
90 sec
120 sec
150 sec
180 sec
210 sec
240 sec
270 sec
300 sec
330 sec
360 sec
390 sec
420 sec
450 sec
480 sec
510 sec
540 sec
570 sec
600 sec

Week 2: Measure effectiveness of neem on egg laying

We used the plates set up the previous week to determine whether the neem affected bean selection by females for egg-laying.

1. Students retrieved their plates from Week 1.

   IF ADULTS WERE NOT REMOVED THEN STEPS 2-5 WERE FOLLOWED. IF ADULTS WERE REMOVED WITHIN 24-48 HOURS THEN SKIP TO STEP 6.

2. They carefully observed the number of surviving adults and dead adults. Note that the elapsed time from the start of the experiment to the observations on adult survival should be less than 14 days since the normal adult life span is approximately two weeks.
3. Using a sorting brush they placed surviving adults into a tube and capped it. Dead adults were placed in a separate tube and capped. They labeled these tubes "live" and "dead" along with their group name on each.
4. They recorded the number of live/dead male and female beetles retrieved from each plate on the data sheet.
5. Each group was given 2 fresh large petri dishes. They labeled 1 plate ìcontrolî and the other plate ìneemî along with their group identity.
6. They carefully removed each bean from the control side of their original plate and observed under the microscope for the presence of eggs. As each bean was analyzed, it was placed into the fresh plate labeled ìcontrolî. They repeated with the beans on the neem side, but placed these in the fresh plate labeled ìneemî. They recorded all observations on the data sheet.

Sample Data Sheet-egg laying and adult survival

	Total # of Beans	Total # of beans with eggs	Total # of beans w/out eggs	# of Live Females	# of Dead Females	# of Live Males	# of Dead Males
Control
Neem

Subsequent Weeks: Observe plates for egg viability. Is there a difference between the neem-exposed eggs and the control?

Experimental Design

Our students readily participated in this exercise and anecdotal evidence suggested they enjoyed it and benefited from the inquiry-based experience.

Unfortunately, we did not see any striking differences in attracting the bean beetles between the compartment that contained beans treated with neem powder and beans that were not treated (see sample data below). However, the exercise was beneficial because:

1.  students searched data bases and each one brought two articles related to either the bean beetle or neem to the class for discussion;
2.  although they had some concept of an 'hypothesis' they did not know how to exactly arrive at it based on prior knowledge and the class discussion related to this exercise helped them to learn and postulate their hypothesis.
3.  students came up with their own experimental designs and each of the five decided-upon designs were discussed in the class for their merits and problems
4.  students were comfortable and very enthusiastic in critiquing ideas of each other
5.  they arrived at an experimental plan (with instructor guidance)
6.  as it was 'their' idea and design, they stayed late in the lab to follow the beetles in the dish
7.  they learned how to pool the data and a bit more about basic statistical analysis.
8.  they also learned how to organize and write a good lab report along the directions of writing a manuscript.

Alternative Experimental Designs: Adult beetle affiliation data could be collected from a Y-maze type apparatus in which beetles had to choose either the neem or an untreated control. Isolating the two treatments (not in the same dish) might provide clearer discrimination results. However, a maze would not be necessary to conduct experiments on egg laying, egg viability, or adult survival. Those studies could be conducted with completely separate dishes, some containing neem and others untreated. Studies on adult survival should be conducted during a period of less than 14 days since the normal adult life span is approximately two weeks.

Data Collection

See the sample data sheets provided above for the described experimental design. Depending on the size of the lab several replicates of each condition may be conducted. In our labs we had 3-5 replicates. Behavioral data should be collected on the same day as the initiation of the experiment. Adult beetles need to be removed within 24-48 hours after initiation and oviposition checked soon thereafter. Egg viability could be recorded 4-6 weeks after oviposition.

Data Analysis

See sample data sheets provided. If students have no prior statistical experience then calculation of pooled totals (for instance, total migrations to neem vs control side) is simple and hand-drawn graphs are appropriate. For more advance groups, Chi-Square tests for the pooled frequency data (total beans in neem and control), are recommended.

Equipment and supplies

Students can be arranged in groups from 3 (recommended) up to 5. The following is the supply list for a class of 30:

20-30 male bean beetles                                          neem leaf powder or neem oil

30-50 female bean beetles                                       3-5 spray bottles (if using Neem oil)

10 large petri dishes (150mm)                                 10 magnifier or dissecting microscopes

10 small petri dishes (35mm)                                   cardboard strips to make dividers

mung or other desirable beans                                 tape to secure cardboard dividers

10 sorting brushes                                                    10 plastic zipper bags

10 marking pens                                                       liquid detergent (if using Neem oil)

 

Supplier of neem

NEEM Leaf Powder

http://www.nextag.com/neem-powder/shop-html or VitaminMegaShop.com

Neem Leaf Powder (Certified Organic) - 1 lb azadirachia indica

1 lb

$21.67

Neem Oil

http://www.organeem.com/    Follow mixing directions on bottle.

Acknowledgments

Some information for this lab was obtained from A Handbook on Bean Beetles, Callosobruchus maculatus by Christopher W. Beck and Lawrence S. Blumer, 2011 (www.beanbeetles.org). We also thank Dr. Anne Grippo and Ms. Amanda Lovelace for assisting in the development and execution of the Bean Beetle lab exercises.

 

Copyright © by Malathi Srivatsan and Amy R. Pearce, 2013. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.

Our students did not have any statistics background, so we did not collate actual numerical migration data from the students, rather, it was presented in a descriptive manner (Table 1). Unfortunately, we did not see any consistent or striking difference in deterring or attracting the bean beetles between the compartment that contained beans treated with neem and untreated beans. However, the 0.75 g group showed some possible deterrent property that should be explored further (Figure 1).

Table 1. Bean beetle (Callosobruchus maculatus) movement between control (C) and neem (N) treated mung beans (Vigna radiata). The total mass of beans on each side of the test petri dish was 17.0g and number of beans of each side of the test petri dish varied from 1185 to 1553. The treatment groups were a negative control (Neg C, no neem applied), 0.50g, 0.75g, and 1.00g neem powder applied to the beans on the neem side, and a positive control (Pos C, the same amount of neem applied to both sides). A total of five replicates were conducted for each treatment with 3 male and 2 female beetles. Movements between the two sides of each dish were monitored every 30 seconds for 10 minutes. The movements of male and female bean beetles were characterized in categories, but a clear pattern did not emerge. Survival after a two week period is shown for the 15 males and 10 females used per treatment group. Note that very few individual beetles survived in the test apparatus (a divided petri dish) for two weeks so survival data were not informative. Future studies on adult survival in this type of experiment might use a shorter exposure period prior to evaluating survival rates, since the normal adult life span is approximately two weeks. Furthermore, adult survival studies might have yielded different results if separate replicate dishes of either a control or neem treatments were employed rather than split dishes.

	Migrations to Each Side		Male live		Female live
Group	C	N	C	N	C	N
Neg C	+	+	0	0	0	0
0.50 g	-	+	0	1	1	1
0.75 g	-	-	1	1	3	0
1.00 g	-		0	1	0	0
Pos C	+	+	1	1	1	0
"-" signifies that beetles did not move at all to that side
no term signifies beetles moved just a few times to that side
"+" signifies beetles moved often to that side
"++" signifies beetles moved very actively to that side, more than "+"

Figure 1. The percentage of mung beans (Vigna radiata) that received bean beetle eggs in the control and neem sides of test petri dish for each of five treatments groups is shown. The total mass of beans on each side of the test petri dish was 17.0g and number of beans of each side of the test petri dish varied from 1185 to 1553. The treatment groups were a negative control (Neg C, no neem applied), 0.50g, 0.75g, and 1.00g neem powder applied to the beans on the neem side, and a positive control (Pos C, the same amount of neem applied to both sides). There were no significant differences between treatment groups. The egg distribution data were collected one week after the start of the experiment. A total of five replicates were conducted for each treatment group and each replicate contained 3 male and 2 female beetles.

Also, after two attempts at exposing the beetles to beans coated with oil, we realized the property of the oil itself was a deterrent simply because the beetles would get stuck in it. We realized that to use the oil, the beans must be either soaked and allowed to dry or sprayed and the remaining residue removed. Beyond this, we did not systematically study the application of Neem oil, although it may be more promising. Refer to the Instructor's Notes on the benefits of this exercise. The raw data are available in the Downloads section along with a powerpoint slide of Figure 1.

Student Handout [pdf] [doc]

Instructor's Notes [pdf] [doc]

Sample data [xls]

Sample data graph [ppt]

Data collection forms [doc] [pdf]

Laboratory writing assignment [doc] [pdf]

Identifying the sexes [ppt]

Egg on bean [ppt]

Test apparatus [ppt]