Background and Hypotheses:
Recent studies, most notably Gbaye et al. (2011, 2012), have investigated the sensitivity of bean beetles in the genus Callosobruchus to organophosphate insecticides (OPs). Economically this is important work given that these beetles are pests that threaten agricultural yields of legumes. OPs work by inhibiting the enzyme acetylcholinerase (AChE) in the beetles. Without this enzyme, the neurotransmitter acetylcholine can’t be broken down and its overflow leads to the eventual death of the beetle. Results obtained by Gbaye et al. (2011, 2012) suggest that there are indeed differences in AChE sensitivity to malaoxon – a popularly used OP – amongst different geographical strains of Callosobruchus maculatus, one particular species of Callosobruchus. This difference may be due to environmental factors such as temperature and food source. In our study, we asked whether the food source (e.g. type of bean) will affect the sensitivity of the beetles’ AChE to malaoxon. Our alternative hypothesis was that AChE extracted from beetles of the species Callosobruchus maculatus will exhibit different sensitivity to malaoxon depending on whether they were bred on mung beans (Vigna radiata) or black-eye peas (Vigna unguiculata). Our null hypothesis, then, was that there will be no such difference in sensitivity to malaoxon.
To test this hypothesis, we set up a manipulative experiment attempting to isolate the effect of the food source on the sensitivity of AChE in C. maculatus to malaoxon. Our independent variable was the type of bean on which the beetles were bred. We required two treatment levels: one using beetles bred on mung beans and the other using beetles bred on black-eye peas. There is no control group in the experiment. However, we performed an enzyme assay (explained below) on controlled ‘blanks’ where no inhibitor was present in order to calibrate the calculated relative inhibition (also explained below). Our sample size was eight for the mung bean experimental group, and the sample size was seven for the black-eye peas experimental group.1 Our dependent variable was the relative inhibition (%) of AChE, thereby indicating sensitivity to malaoxon. We attempted to standardize as many variables as possible. Differently than Gbaye et al. (2012), our goal was only to isolate the effect of the food source so we standardized the species of beetle: C. maculatus, an LB strain collected by Rodger Mitchell from Ohio State University. (The geographical strain of the beetles is not known.) We also standardized the concentration of malaoxon added during the assay.
Beetles from both groups were cultured concurrently, so the ages and maturities of the beetles are presumably standardized. We performed a colorimetric enzyme assay on both experimental groups at room temperature. Perhaps most importantly, the same procedure for the enzyme assay was performed on both experimental groups. Our experiment had only one replication but can and should be repeated. The enzyme assay basically works like so: we will let the AChE enzyme present in each beetle react in vitro with ATCI (not the usual substrate ACh present in the beetles, though the difference is presumably negligible). The ATCI is hydrolyzed to produce acetate and thiocholine. Thiocholine in particular will react with an indicator compound DTNB to form TNB-. When present in water, this ionizes further to TNB2-. This compound shows as yellow and we can detect its relative presence using a spectrophotometer.
The lesser the yellow absorbance in the product, the greater the sensitivity to malaoxon as measured by relative inhibition. The relative inhibition is the ratio of the observed difference in absorbances between the blank and experimental trials with to the absorbance in just the blank. (Of course multiplying by 100 yields relative inhibition as a percentage.) The specific procedure and materials used is found on pp. 73-76 of (Course Supplement, 2014). Once we collected the data on the 15 individual beetles, we found the sample mean relative inhibitions per experimental group and their respective sample standard deviations. We then used a two-tailed t-test analysis to determine if the difference in means is statistically significant. Our experimental prediction: if we perform the colorimetric enzyme assay (described on pp. 73-76 of the Course Supplement) on two groups of beatles – one cultured on mung beans and one cultured on black-eye peas – we will observe a statistical difference in the respective relative inhibitions.
Results and Data Analysis:
Beetles bred on mung beans exhibited a mean relative inhibition of 35.32% with a sample standard deviation of 26.41%. Beetles bred on black-eyed peas exhibited a mean relative inhibition of 35.42% with a sample standard deviation of 17.97%. This data is reflected in the bar graph in Figure 1 below. The large bars show the respective means and the error bars show a range of one standard deviation about the mean. A two-tailed t-test yields a t-value of 0.99. For 13 degrees of freedom, t-critical is 2.16 for a 95% confidence level and 1.35 for an 80% confidence level. This is reflected in Table 1 below. Figure 1: Mean relative inhibition, as a function of the type of bean on which the beetles were bred. The error bars represent one standard deviation.
Table 1: Results of two-tailed t-test for the comparison of mean relative inhibition.
Since t-critical for a 95% confidence level far exceeds our calculated t-value of 0.99, we cannot be confident that the difference in means is statistically significant to this level. What’s more, our t-value doesn’t reach t-critical for even a 80% confidence interval, meaning there is at least a 20% likelihood that the observed difference in means is due to random variation.
Conclusions and Discussion:
A 20% chance that the difference in means is due to random variation is far too great to support the alternative hypothesis – that there is a difference in sensitivity of AChE in C. maculatus to malaoxon bred on mung beans as opposed to black eyed peas. By default our data supports the null hypothesis – that there is no such difference in sensitivity. However, we don’t feel we have yet proven this negative fact. Importantly, we are not aware of what geographical strain of beetle we used in this experiment. Gbaye et al. (2012) observed a statistical difference in AChE activity between C. maculatus beetles bred on mung beans and cowpeas (i.e. black-eyed peas) amongst all three geographical strains tested – those from Cameroon, Brazil, and Yemen. However, the difference in the Cameroon strain was least apparent. This suggests that our strain of beetle may have been from either Cameroon or another location not tested in Gbaye et al. Our experiment should be replicated using beetles of a known geographic strain for comparison. Also, our sample sizes were presumably much smaller than theirs – though Gbaye et al. only specify that they used “two populations reared separately on cowpea … and mungbean” – and so our experiment should be replicated using a much larger sample size, making it easier for a t-test to detect a statistical difference in the mean relative inhibitions.
Further, we investigated only the C. maculatus species of bean beetle, which in Gbaye et al. exhibited a smaller effect of food source on AChE activity than either C. chinensis or C. rhodesianus. Our experiment, when replicated, should increase the treatment levels three-fold (or more) accounting for investigations on these two (or more) additional species within the genus. It would also be useful to investigate the effect of other food sources (e.g. other legumes) of relative inhibition of AChE. We finally suggest that further research include the effects of other OPs besides malaoxon to determine the best course of action for legume farmers to limit the infestation of beat beetle pests. If in the course of this suggested research we still are at a loss to find a statistical difference, environmental factors other than food source should be tested for their effect on AChE relative inhibition. Knowing the effect of temperature could provide farmers with information on ideal climates in which to grow their crops to limit pest infestation. To test this an experiment could performed using the same procedure as above and include a manifold of treatment levels, testing beetles bred at temperature regimes ranging from cold (e.g. 15 degrees) to hot (e.g. 40 degrees). Of course in that case, a simple t-test cannot be performed.
Course Supplement for Biological Foundations I Bio 10100, 2014. Department of Biology City College of New York.
Gbaye, O. A., Millard, J. C., & Holloway, G. J. (2011). Legume type and temperature effects on the toxicity of insecticide to the genus Callosobruchus(Coleoptera: Bruchidae). Journal of Stored Products Research,
Gbaye, O. A., Holloway, G. J., & Callaghan, A. (2012). Variation in the sensitivity of Callosobruchus (Coleoptera: Bruchidae) acetylcholinesterase to the organophosphate insecticide malaoxon: effect of species, geographical strain and food type. Pest management science, 68(9), 1265-1271.
1Note that we experienced technical difficulties in our investigation of one of the beetles in the mung bean experimental group, where the presence of AChE thus accounting for the difference in the sample size.