Adapted from L. Blumer, Morehouse University and C. Beck, Emory University
Preparation for the Laboratory: Read the description of bean beetles and their background from the Moodle site; complete Tutorial XX in Moodle.
Bean beetles (Callosobruchus maculatus) are agricultural pest insects of Africa and Asia. Females lay their eggs on the surface of beans (Family: Fabaceae). Eggs are deposited singly and several days after oviposition, a beetle larva burrows into the bean. Larval growth and pupation occur inside the bean and are consequently difficult to observe. At 30°C, pupation and emergence of an adult beetle occurs 25-30 days after an egg is deposited (Blumer and Beck, 2008). Adults are mature 24-36 hours after emergence, and they do not need to feed as adults. Adults may live for 7-10 days during which time mating and oviposition occur. Since larvae cannot move from the bean on which the egg was deposited, the oviposition choice of a female determines the future food resources available to her offspring. The choice of prey bean is the most critical choice a female makes for her offspring, as it will influence their growth, survival and future reproduction (Mitchell, 1975; Wasserman and Futuyma, 1981).
Female beetles are easily identified in the live cultures because of the two dark stripes on the posterior of the abdomen. The posterior abdomen of the male is uniformly light in color (Figure 1).
Figure 1. Dorsal view of male (left) and female (right) bean beetles. The sex-specific coloration of the posterior abdominal plate (pygidium) is shown (Brown and Downhower, 1988)
In class you will be provided with live bean beetle cultures and supplies of different kinds of dried beans. The seeds available for this experiment will include: mung beans (Vigna radiata), black eyed peas (cowpea) (Vigna unguiculata), garbanzo (Cicer arietinum), kidney, pinto, and black beans (Phaseolus vulgaris), soy beans (Glycine max), adzuki beans (Vigna angularis), lima beans (sieva bean) (Phaseolus lunatus), and green pea (Pisum sativum).
Beetles can be moved using either sorting brushes or soft forceps. When removing beetles from the stock cultures, Petri dishes or plates, lightly tap the container on the lab bench before removing the lid to keep beetles from crawling out immediately. If the lid is left off for more than a minute or so, the beetles will escape from the culture container. Please do not let any beetles escape! Although they are not agricultural pests in North America, it is good scientific practice to keep experimental organisms contained within the laboratory.
Since the oviposition choices of females influence the survival and reproductive success of their offspring, females may be very sensitive to the species and condition of the beans on which they deposit their eggs.
As groups, discuss what factors might determine whether bean beetle eggs are laid on a particular variety of bean, and how you would design an experiment to test whether female bean beetles discriminate among bean species.
Remember, we are not asking you to design an experiment to figure out WHAT makes the beans more or less preferable, but rather to design an experiment to determine if there is a preference at all.
As a class we will come up with an experimental design that will test our hypothesis. Once we have determined what the experimental design will be, we will set up our experiment and do our analysis at the next class time. Please record in your laboratory notebook the protocol that we are following before you leave class this week.
Read the Swella and Mushobozy (2009) paper that is on the Moodle site.
Read the Pechenik writing guide section that pertains to writing about statistics (pp. 64-67). Compare your writing to the examples set in this chapter- do you remember to write about the biological question and not focus on the statistical results?
References:
Blumer, L. S. and Beck, C.W. 2008. Oviposition substrate choice by bean beetles, Callosobruchus maculatus. Association for Biology Laboratory Education (ABLE) 2007 Proceedings, Vol. 29:51-66
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 Fabrivius (Coleoptera: Bruchidae). Evolution 35:605-617.
Preparation for Laboratory: Read the Swella and Mushobozy (2009) paper from the Moodle site. Read indicated selections from Pechenik writing guide on statistics and the Discussion section
Last week, we implemented our experimental design. The bean beetles have been reserved in their Petri dishes, and this week we will be counting the number of eggs on each bean type. Once you have collected the data for your group, we will collect the data for the whole class.
Before we do this, however, we need to learn about a new type of statistical test that may help us understand our results.
Are there more eggs on one particular type of bean than another?
An appropriate test to use is the Chi-Squared test (Χ2), because the type of data we are collecting are categorical data (also called qualitative data). In other words, each bean type falls into a particular group. For each bean type, we have a count or a frequency for the number of eggs that were laid on that type of bean. This count can be expressed as a percentage or as a simple count of the total individuals that fall into that category.
The Chi Squared test determines the expected count for each category based on the assumption that there is no difference between the treatments (i.e. lima beans vs. black beans). Then it assesses whether the observed pattern is significantly different by calculating the Chi-square statistic and comparing it to a critical value:
Open up the class data from the class file- your instructor will tell you what the file string will be. The easiest way to do this is by pressing the “Import Dataset” in the upper right hand window- you will be prompted to select whether you are getting the data from the web or from a file.
The screen will read something like this:
Bean.Beetle.Data <- read.csv(“/Volumes/public/Data/BIOLOGY/R Workshop Datasets/Bean Beetle Data.csv”)
View(Bean.Beetle.Data)
Now you have the dataset loaded onto R. You will be able to see your dataset in a window on the lefthand side of your screen, and can begin to understand the Results of our class. Note that each group’s data are still separate from each other. Let’s take a look at what the total number of eggs laid on each species is:
sum(Count~Species, data=Bean.Beetle.Data)
## P._vulgaris_(black) P._vulgaris_(kidney) P._vulgaris_(navy)
## 75 52 138
## P._vulgaris_(pinto) Vigna_angularis Vigna_radiata
## 197 136 106
## Vigna_unguiculata
## 203
You will see the total number of eggs laid for each bean species. Let’s rename these Results to something simple, like “Results”:
Results=sum(Count~Species, data=Bean.Beetle.Data)
Now, let's take a look at our data. Since we are only interested right now in total counts, we can graph everything in a bar chart.
barplot(Results)
What does your graph tell you about egg deposition? Is it random, or is there a preference? Is your graph the best representation, or do you wish to modify it?
Now, let's see what the Chi-squared test tells us about female bean beetle oviposition preference:
chisq.test (Results)
##
## Chi-squared test for given probabilities
##
## data: Results
## X-squared = 151.3, df = 6, p-value < 2.2e-16
You will see the X-squared test result, the degrees of freedom (which is related to the total number of samples), and a p-value. Don't worry about what the p-value means right now.
References:
Brown, L. and Downhower, J.F. 1988. Analyses in Behavioral Ecology: A Manual for Lab and Field. Sinauer Associates, 194 pp.
Wasserman, S.S. and D.J. Futuyma. 1981. Evolution of host plant utilization in laboratory populations of the southern cowpea weevil, Callosobruchus maculatus Fabrivius (Coleoptera: Bruchidae). Evolution 35:605-61
Swella, G.B. and Mushobozy, D.M.K. 2009. Comparative susceptibility of different legume seeds to infestation by cowpea bruchid Callosobruchus maculatus (F.). Plant Protection Sci. 45 (1): 19-24.