Chelsy Sagastume
Marlena Osei, Monique Duffus, Star Gonzalez

The Effect of Food Source on Enzyme Activity In Bean Beetles

Callosobruchus maculatus is a species of beetles also known as bean beetles. Found in tropical and subtropical locations, such as the regions of Africa and Asia (Course Supplement, 2014), the larvae of the bean beetles feed and evolve on mung beans. Mung beans are a plant species in the legume family. While mung beans provide many health benefits to humans, bean beetles become pests to mung beans as soon as the larvae start to evolve. Adult females lay fertilized eggs on the bean seed, from there, once the larvae hatches from the egg, it burrows from egg through the seed coat and into the bean endosperm. The larvae ultimately feeds on the endosperm and embryo and destroys the bean crop. The solution for reducing and controlling the infestation of Callosobruchus maculatus, is the use of organophosphate insecticides such as malaoxon. The insecticide works by acting as an inhibitor, damaging an enzyme in the body called acetylcholinesterase (AChE). This enzyme is crucial for regulating nerve signals. AChE is in charge of breaking down the neurotransmitter acetylcholine and keeping it at a concentration suitable for the body. If this enzyme in the bean beetle is damaged, in this case by an insecticide, then it could lead to their demise. However the bean beetles are able to develop resistance to the insecticide, thus the Callosobruchus maculatus that survive have a higher detoxification systems that directly affect the tolerance to the insecticide.
Our class experiment focused on the effect of different food sources on the level of activity of detoxification enzymes in bean beetles. During our examination we compared the activity levels between two groups of bean beetles that were cultivated on two different types of seeds used as a food source; adzuki and mung beans. Our alternative hypothesis is, there will be a significant difference in the mean of the levels of enzyme activity for the bean beetles developed on two different food sources. The null hypothesis is, there will not be a significant difference in the mean of the levels of enzyme activity for the bean beetles developed on two different food sources.

Experimental Design
Our experimental design was formulated by one experiment in which we used two treatment levels with different substrates-- alpha-naphthyl acetate or beta-naphthyl acetate. The goal in this experiment is to measure the activity level of each enzyme, therefore the two different substrates will be hydrolyzed to get products alpha-naphthol or beta-naphthol. These products will mix with the dye given, Fast Blue B Salt . The Fast Blue B Salt is used for the determination of acetylcholinesterase activity, therefore causing a change in absorbance of light, this will then be measured using a spectrophotometer. The absorbance of the solution permits us to measure the activity level of each enzyme. Our food sources adzuki and mung seeds remained as our independent variables. Our dependent variable is the level of enzymatic activity. In this experiment we required two experimental treatment levels, one with the food source as adzuki beans and the other with mung beans as the food source. There was no control group in this experiment. There was only one replication per group. The sample size of our group experiment was 4 in each treatment level, therefore 8 beetles in total. The sample size of the class data was 14 beetles for the Adzuki bean and 15 for the mung bean. The goal of this experiment is to recognize the effect of food on levels of enzyme activity of bean beetles, therefore we standardized the type of species used for this study; Callosobruchus maculatus bean beetles. These beetles come from the same strain, which is also a standardized variable in this experiment, LB strain.
The experiment started with observing the sex of the beetles in each treatment level. Treatment 1 contained two males and two females. Treatment 2 contained two males and two females as well. We put the bean beetles tissues and the homogenizing buffer to each tube using a micropipettor to get the correct measurement of product in the tubes. After this step we transferred the supernatant from