Bioprotection from Beetles: Investigating the Untapped Secrets of the Neem Tree (Azadirachta indica) Instructor s Notes

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1 Bioprotection from Beetles: Investigating the Untapped Secrets of the Neem Tree (Azadirachta indica) Instructor s Notes 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 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). 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 bean 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. 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

2 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 28 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 writing assignment. 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 hours of placement. Discrimination tests of 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.

3 Sample Data Sheet adult location preference Zero Time: Time 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 # of Migrations to Control Side # of Migrations to Neem Side

4 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 HOURS THEN SKIP TO STEP They carefully observed the number of surviving adults and dead adults. 4. 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. 5. They recorded the number of live/dead male and female beetles retrieved from each plate on the data sheet. 6. Each group was given 2 fresh large petri dishes. They labeled 1 plate control and the other plate neem along with their group identity. 7. 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 control. They repeated with the beans on the neem side, but placed these in the fresh plate labeled neem. They recorded all observations on the data sheet. Sample Data Sheet egg laying and adult survival Control 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 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.

5 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). 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 or egg viability. Those studies could be conducted with completely separate dishes, some containing neem and others untreated. 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 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

6 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 Supplies for a class of 30 students male bean beetles neem leaf powder or neem oil 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 eem-powder/shop-html or VitaminMegaShop.com Neem Leaf Powder (Certified Organic) - 1 lb azadirachia indica 1 lb $21.67 Neem Oil Follow mixing directions on bottle. ITEM PRICE (US$) CPNO % Pure Cold-pressed Neem Oil (2 oz. Bottle) $ 4.99 CPNO % Pure Cold-pressed Neem Oil (8 oz. Bottle) $ CPNO % Pure Cold-pressed Neem Oil (32 oz. Bottle) $ CPNO-1G 100% Pure Cold-pressed Neem Oil (1 Gallon Jug) $ 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 ( We also thank Dr. Anne Grippo and Ms. Amanda Lovelace for assisting in the development and execution of the Bean Beetle lab exercises.