Sarah Alsilwi
Rene Kokoszka, Tahsin Khan, John Vlahos


Examining the effects of Mung Beans and Black Eyed Peas on Malaoxon induced inhibition of AChE

Introduction:

Callosobruchus Maculatus, otherwise known as bean beetles, are part of the subfamily Bruchinae. Bean beetles are insects or crop pests found in tropical regions of Africa and Asia (CCNY Lab Manual, 2004). They lay eggs on the surface of bean seeds such as mung beans (vigna radiata) or black eyed peas (vigna unguiculata), hence their name, bean beetles. C. maculatus don't require water or food in their short life-span of 10-14 days1.The larvae of the beetle feeds on the endosperm and embryo of the seed, destroying the bean crop. Ways to control or eliminate these pests are through organophosphate insecticides such as Malaoxon. This insecticide inhibits the enzyme acetylcholinesterase (AChE), which is an important part of cell-cell signaling in the nervous system. Interfering with this process of breaking down the neurotransmitter impedes the correct functioning of the nervous system, eventually killing the pest. However, insects have grown susceptible to the insecticides showing resistance through detoxification of the chemicals used to kill them. The food source of the insect plays a role in this resistance by transferring defensive chemicals from the plant to the beetle. However, the main question is whether different food sources, like mung beans and black eyed peas, affect the level of activity of the detoxification enzymes in bean beetles.
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1 Nicholls, Stu. "A Handbook on Bean Beetles, Callosobruchus maculatus." BeanBeetles.org/A Handbook on Bean Beetles: Laboratory Methods. N.p., n.d. Web. 22 Apr. 2017.

We hypothesized that the bean beetles' alpha-naphthyl acetate esterase (ANAE) and beta-naphthyl acetate esterase (BNAE) enzyme activity will be different depending on the food source, mung beans and black eyed peas. The null hypothesis was that the beetles' alpha-naphthyl acetate esterase (ANAE) and beta-naphthyl acetate esterase (BNAE) enzyme activity will not be different depending on the food source, mung beans and black-eyed peas. The purpose of this experiment is to understand the relationship between pests and their food sources, along with further understanding the resistance in pests to insecticides.

Methods -
As stated in the CCNY Lab Manual, 2004, the methods to complete this experiment are as follows. For the beetles grown on mung beans, crude extracts were made to separate the enzymes from the bean beetles and add them to different substrates. 36 crude extracts made for each substrate, the alpha-naphthol actetate and beta-naphthyl acetate, to produce different products. After adding the homogenizing buffer to the beetles in the microcentrifuge tubes and inverting five times, the tubes were placed in a centrifuge for several minutes to get the protein of the beetles. The tubes were vortexed to simply get rid of bubbles. Then the crude extracts were left to incubate to speed up the reaction or to cause collisions. After incubation, Fast Blue B Salt Stain solution was added to each tube to stain the enzymes and proteins, then incubating the tubes once more. After incubation, the supernatants are transferred to cuvettes which were placed in the spectrophotometer at 595 nm to read the light absorptions of all the solutions. Blank tubes that contained 300 ul of homogenizing buffer and 240 ul of either alpha or beta acetate solution, were also made and placed in the spectrophotometer simply to calibrate the spectrophotometer. After placing the labelled cuvettes and blank cuvettes into the spectrophotometer, the absorbance readings were read at 595 nm to calculate the protein concentration of all the tubes which was used for the other methods of data analysis.

Experimental Design-
The independent variables are the mung beans and black eyed peas or food source of the insects, since we intentionally altered the food source to see its effect on bean beetles. The dependent variable is the detoxifying enzyme activity of both the beetles grown on mung beans and black eyed peas. The standardized variables are the amount homogenizing buffer added, the time for tubes to centrifuge, the time for tubes to be vortexed, the blank tubes, incubation times, the amount of alpha-naphthyl acetate and beta-naphthyl solution added, the amount of BSA stock solution added to each cuvette, the temperature in which the tubes were kept, the spectrophotometer, and the amount