Adapted from C. Beck and L. Blumer by Staci Forgey, TCC Biology Faculty

Introduction

Bean beetles, Callosobruchus maculatus, are herbivorous pest insects that are found in Africa and Asia. Females lay their eggs on the surface of beans. Eggs are layed singly, and hatch into larvae (maggots) several days later. The larva then burrows into the bean and will form a pupa 21–30 days after the egg was deposited. They mature 24–36 hours after emergence from the pupa and do not need to feed.

Adults typically live for 1–2 weeks. Mating and oviposition occur during this time period.  Females will choose the best substrate (bean) to lay their offspring on, since the larvae cannot move. By choosing a substrate for oviposition, the female chooses the food resource available to her offspring (Brown and Downhower 1988). This is a critical choice for the female, as it influences the growth, survival and future reproductive success of her offspring (Mitchell, 1975, Wasserman and Futuyma, 1981). Females can lay eggs on a wide range of bean species, but very few bean species will result in normal development and the successful emergence of adults. Some species of beans are toxic to larvae (Janzen 1977). 

Materials

In class, you will be provided with live cultures of bean beetles containing adults that have been raised on mung beans (Vigna radiata).

Female beetles are easily identified in the live cultures because they have two dark stripes on the posterior of the abdomen, whereas the posterior abdomen of males is uniformly light in color.

You will also have access to petri dishes and several types of beans. 

Experimental Design

Since the oviposition choices of females influences the survival and future success of their offspring, females may be very sensitive to the species and condition of the beans on which they are depositing eggs.

Each group should design a set of experiments to address whether female bean beetles discriminate between suitable species of beans. We will re-visit the experiment next week and collect our data set.  We will choose one experiment to perform as a class. 

As a group, list several ways we might examine female bean choice for oviposition.  We will pick one experiment from the class to perform.  Write your ideas below.

**Stop here. We will go over ideas for our experiment as a class and choose one to perform**

Outline the agreed on experiment using the criteria below. 

1. Formulate a hypothesis for this week’s experiment. Be specific!
2. We will re-visit the experiment in a week to collect data on the number of eggs laid.  Formulate a hypothesis for this experiment.    
3. Identify the independent variable(s).
4. Identify the dependent variable(s).
5. What variables will you keep standard?
6. What is your control?
7. Design data collection tables for both of your experiments on a separate sheet of paper.

For this data, it is most useful to perform a Chi Square analysis. Chi Square statistical test evaluate whether this is a significant difference between groups of data. The null hypothesis (H₀) is the hypothesis that states that there is no difference between the groups of data. The alternative hypothesis, (H_a) is the hypothesis you outlined above.

1. Restate your H₀ hypothesis.
2. H_a hypothesis.
3. If the null hypothesis is correct, what would we expect to see?
4. If our alternative hypothesis is correct, what would we expect to see?

To do this statistical test, we need to calculate observed and expected values for the bean species and for our control.  Our expected values are based on our null hypothesis (that there is no difference).

For example, if we had 40 eggs and four different treatment groups, we would expect there to be 10 eggs on each group. 

Treatment	Observed number of eggs	Expected number of eggs
Mung beans	5	10
Pinto beans	25	10
Chick peas	10	10
Control	0	10

Fill in the data table below with our observed (what we actually found) and expected values. 

Treatment	Observed number of eggs	Expected number of eggs

To calculate the chi-square value, or χ ² , we simply add the square differences, divided by the expected, of all the observed and expected.  In mathematical terms:

[latex]\displaystyle\chi^2=\Sigma\frac{(O-E)^2}{E}[/latex]

So for our example from the previous sample table, the first [latex]\displaystyle\frac{(O-E)^2}{E}[/latex] would be [latex]\displaystyle\frac{(5-10)^2}{10}=2.5[/latex] 

We would then add to this value all of the other[latex]\displaystyle\frac{(O-E)^2}{E}[/latex] in the table and then add to get the[latex]\displaystyle\chi^2[/latex]  value. 

Treatment	Observed number of eggs	Expected number of eggs	Observed-Expected	(Observed-Expected)2	(Observed-Expected)2/Expected

Compute the χ² value for your data by adding all of the (Observed-Expected)2/Expected values.

In order to find something to compare this number with, we need to calculate the degrees of freedom, or the number of different comparisons that can be made within the table. The degrees of freedom are the number of columns (M) minus one times the number of rows (N) minus one.

Degrees of freedom = (M − 1)(N − 1)

A	B
C	D

How many ways can this two by two table be broken into individual comparisons?  Hint:  Use the formula above. 

Calculate the degrees of freedom in your experiment. 

Now we can compare against the Chi distribution for the likelihood that our data is generated by chance. Remember, we are looking at the column labeled 0.05 for our p value.  This means that at this level, we are 95% sure our results are real. The Chi distribution table gives us a critical value to compare our test value to. 

• If test > critical value @ p level, reject null hypothesis
• If test < critical value @ alpha level, fail to reject null hypothesis

Compare to the Chi-distribution table. Did your results come about by chance?

DF/P	0.995	.990	0.975	.950	.900	.750	.500	.250	.100	.050	.025	.010	.005
1	0.00004	.00016	0.001	0.004	0.016	0.102	0.455	1.323	2.706	3.841	5.024	6.635	7.879
2	0.010	0.020	0.0506	0.103	0.211	0.575	1.386	2.773	4.605	5.991	7.378	9.210	10.597
3	0.072	0.115	0.216	0.351	0.584	1.213	2.366	4.108	6.251	7.815	9.348	11.345	12.838
4	0.207	0.297	0.484	0.711	1.064	1.923	3.357	5.385	7.779	9.488	11.143	13.277	14.860
5	0.412	0.554	0.831	1.145	1.610	2.675	4.351	6.626	9.236	11.070	12.833	15.086	16.750
6	0.676	0.872	1.237	1.635	2.204	3.455	5.348	7.841	10.645	12.592	14.449	16.812	18.548
7	0.989	1.239	1.690	2.167	2.833	4.255	6.346	9.037	12.017	14.067	16.013	18.475	20.278
8	1.344	1.647	2.180	2.733	3.490	5.071	7.344	10.219	13.362	15.507	17.535	20.090	21.955
9	1.735	2.088	2.700	3.325	4.168	5.899	8.343	11.389	14.684	16.919	19.023	21.666	23.589
10	2.156	2.558	3.247	3.940	4.865	6.737	9.342	12.549	15.987	18.307	20.483	23.209	25.188
11	2.603	3.053	3.816	4.575	5.578	7.584	10.341	13.701	17.275	19.675	21.920	24.725	26.757
12	3.074	3.571	4.404	5.226	6.304	8.438	11.340	14.845	18.549	21.026	23.337	26.217	28.300
13	3.565	4.107	5.009	5.892	7.042	9.299	12.340	15.984	19.812	22.362	24.736	27.688	29.819
14	4.075	4.660	5.629	6.571	7.790	10.165	13.339	14.114	21.064	23.685	26.119	29.141	31.319
15	4.601	5.229	6.262	7.261	8.547	11.037	14.339	18.245	22.307	24.996	27.488	30.578	32.801
16	5.142	5.812	6.908	7.962	9.312	11.912	15.339	19.369	23.542	26.296	28.845	32.000	34.267
17	5.697	6.408	7.564	8.672	10.085	12.792	16.338	20.489	24.769	27.587	30.191	33.409	35.718
18	6.265	7.015	8.231	9.390	10.865	13.675	17.338	21.605	25.989	28.869	31.526	34.805	37.156
19	6.844	7.633	8.907	10.117	11.657	14.562	18.338	22.18	27.204	30.144	32.852	36.191	38.582
20	7.434	8.260	9.591	10.851	12.443	15.452	19.337	23.848	28.412	31.410	34.170	37.566	39.997
21	8.034	8.897	10.283	11.591	13.240	16.344	20.337	24.935	29.615	32.671	35.479	38.932	41.401
22	8.643	9.542	10.982	12.338	14.041	17.240	21.337	26.039	30.813	33.924	36.781	40.289	42.796
23	9.260	10.196	11.689	13.091	14.848	18.137	22.337	27.141	32.007	35.172	38.076	41.638	44.181
24	9.886	10.856	12.401	13.848	15.659	19.037	23.337	28.241	33.196	36.415	39.364	42.980	45.559
25	10.520	11.524	13.120	14.611	16.473	19.939	24.337	29.339	34.382	37.652	40.646	44.314	46.928
26	11.160	12.198	13.844	15.379	17.292	20.843	25.336	30.435	35.563	38.885	41.923	45.642	48.290
27	11.808	12.879	14.573	16.151	18.114	21.749	26.336	31.528	36.741	40.113	43.195	46.963	49.645
28	12.461	13.565	15.308	16.928	18.939	22.657	27.336	32.620	37.916	41.337	44.461	48.278	50.993
29	13.121	14.256	16.047	17.708	19.768	23.567	28.336	33.711	39.087	42.557	45.722	49.588	52.336
30	13.787	14.953	16.791	18.493	20.599	24.478	29.336	34.800	40.256	43.773	46.979	50.892	53.672

We will now use this data and information to do a lab write up. 

Lab Report Template

The report should be typed and single spaced. See grading rubric for clarity on formatting.

Title Page

• Should include Title (a brief, concise, yet descriptive title), your name, lab instructor’s name, and lab section.
  • Note: this is a separate sheet

Body of Report

Identify the different sections of the body of the report with headings.

The report should begin with a brief paragraph (complete sentences) that includes a statement of the problem and your hypothesis.  This should be under the heading of Introduction.

• Statement of the problem:
  • What question are you trying to answer?
    
  • Include any preliminary observations or background information about the subject (in this case the bean beetles) such as reproductive cycle, life span etc. Be sure to cite any sources.
• Hypothesis:
  • Write a possible explanation/prediction for the problem/question you are asking.
    
  • Make sure this possible explanation/prediction is a complete sentence and not a question.
    
  • Make sure the statement is testable. In other words, can you perform an experiment that will either support or refute your prediction.  If you cannot not think of a way to test your prediction, then it is not testable.   

Next heading should be Materials:

• Make a list (this does not need to be in paragraph form) of ALL items used in the experiment and their quantities.  Of the materials used, identify which are dependent and independent variables, constants (standardized variable) and control group (you will lose points if you do not identify ALL dependent and independent variables, constants and controls).

Next heading should be Procedure:

• Write a paragraph (complete sentences) which explains what you did in the experiment.
  
• Your procedure should be written so that anyone else could repeat the experiment.  For instance, what beetle did you use? How was your petri dish set up? What beans did you use?   That means that even some of the most obvious steps need to be stated so there is no ambiguity.
• When designing the procedure, be sure to include replicating the experiment to ensure data is reproducible and valid. 

Next heading should be Results:

• Write a paragraph (complete sentences) describing the results and observations of your experiment.  Here you will compare results for controls and variables and not simply list the numbers.
• This section also includes data tables, graphs or charts to illustrate the results of you experiment.  Be sure to include calculated averages of t.
• All tables, graphs and charts should be labeled appropriately (a title, labels for x & y axis, legend etc.) so the reader will be able to understand. 

Next heading should be Conclusions:

• Write a paragraph restating your hypothesis and whether you accept or reject your hypothesis
  
• In this paragraph, explain why you accepted or rejected your hypothesis using data from the experiment. Include a brief summary of the data—averages, highest, lowest, etc., to help the reader understand your results and why you have come to particular conclusions.
• Discuss your thoughts about the possible reasons for your results (for example, if you chose salt water as a variable, give a possible reason why salt water, in particular, may have generated your results). 
  
• Discuss possible errors that could have occurred in the collection of the data (experimental errors) and describe how these errors may have impacted the data.

The following rubric will be used to grade these reports:

	Excellent 5 points	Satisfactory 2.5 points	Unsatisfactory 0 points
Title Page	Contains title, student name, instructor name and section	Missing either instructor name or section	No title page
Formatting: typed, spacing	Typed and single spaced	Typed, but not single spaced	Not typed
Grammar and Spelling	No errors; contains complete sentences and no misspellings	A few minor errors in grammar and spelling	Several major errors in grammar and spelling
Formatting: headings	Each section has a heading as described in template	Some sections lack headings	No headings
Hypothesis	Predictions are clearly stated and written as a testable statement	Predictions/expected outcomes are not clearly stated	Not written as a testable statement
Materials	All equipment and materials described; identify variables, controls and constants	Materials incompletely described	No identification of variables, controls and constants
Procedure	Clear step-by step description	Description missing details making it difficult for another scientist to repeat experiment	Description missing so much detail it would be impossible to repeat
Results	Clearly written description of results comparing controls and variables	Results are presented but no comparison between controls and variables are made	No written description of results
Data tables, graphs or charts	Easy to interpret, clear labels, all data, including calculated averages, included	Disorganized (not easy to understand, missing labels) but all data included	Disorganized and or data clearly missing
Conclusion	Clearly explains acceptance or rejection of hypothesis using data to support conclusion; identifies sources of error	Accepts or rejects hypothesis but does not use data to explain why; or does not identify sources of error	Does not explain conclusion and does not identify sources of error
Total ______out of 50 points

Literature Cited

Brown, L. and J.F. Downhower. 1988. Analyses in Behavioral Ecology: A Manual for Lab and Field. Sinauer Associates, 194 pages.

Janzen, D.H. 1977. How southern cowpea weevil larvae (Bruchidae Callosobruchus maculatus) die on non-host seeds. Ecology 58:921–927.

Mitchell, R. 1975. The evolution of oviposition tactics in the bean weevil, Callosobruchus maculatus F. Ecology 56:696–702.

Wasserman, S.S. and D.J. Futuyma. 1981. Evolution of host plant utilization in laboratory populations of the southern cowpea weevil, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae). Evolution 35:605–617.