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Testing the Effects of Heat Stress on Bean Micro beetle Biome

Diversity

Maura Dianno1, Ben Baumgartner1, Mia Shocket1, George Park1, Caroline Dong1

1Emory University

Objective

The objective is to study the effects for heat stress on bean beetle microbiome diversity.

Figure 2: There is no effect on diversity from heat-stress on a Beetle Microbiome.

The graph shows the average Simpson’s Index for the Non-Stressed Beetle was 1.86 and the average Simpson’s Index for the stressed Beetle is 1.93. The microbiome of both the Stressed and Non-stressed beetles were plated on PEA and EMB plates. The species richness and diversity were recorded and used to find the Average Simpson's index for 146 replicates. The error bars represent +/- 1 of the standard deviation. After the bacteria was counted and the inverse Simpson was found, a two-tailed unpaired T-test was performed. The p-value was found to be 0.624.

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Figure 1: The tables show the unique beetle morphology for the stressed and non-stressed beetle.

The species richness or number of unique species for the stressed beetle is 4 and the species richness for the non-stressed beetle is 3. The inverse Simpson’s for the stressed beetle is 1.68 and the’ inverse Simpson's for the non-stressed beetle is 1.46

Research Approach

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  1. Beetles were heat stressed in 30 degrees Celsius
  2. The exterior of Stressed and Non-stressed Beetles were sterilized and placed in saline solution then crushed, separately to collect interior microbes
  3. The stressed and Non-Stressed Beetles were plated on PEA and EMB plates, sealed with parafilm, and stored for one week at room temperature (20 degrees Celsius)
  4. After one week, colony counts, and unique species morphologies were recorded and used to calculate species richness and inverse Simpson's index
  5. A microbe’s DNA was collected from the plate, and a PCR reaction was performed to amplify and analyze the DNA
  6. Sanger sequencing was performed on the DNA to obtain its genome
  7. BLAST analysis were used to match the microbe genome to the national database.

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Figure 3: Query found in national database that matches microbe DNA. The microbe DNA was sequenced from 90-850. The E value for the match was 0.0, a 98% coverage, and a 97.83% identity.

Conclusions

  • Figure 1 shows the unique beetle morphologies between a non-stressed and a stressed beetle
  • The stressed beetle had a slightly higher species richness than the non-stressed beetle in figure 1
  • In figure 2, the stressed beetles had a higher inverse Simpson’s index than the non-stressed beetles
  • The P value for a two tailed unpaired t-test was 0.624, which means the difference in Simpson’s index is a not a statically significant difference between the heat stressed and non-stressed beetles in figure 2
  • The microbe species studied was matched and identified using the BLAST database

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Future Direction

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The study was very limited to two types of beetles: heat stressed and non heat stressed. An expansion to this project would be to collect beetles from different environments such as dry or temperate and see how they survive in the same conditions.

Acknowledgements

Hana Ye, LA

Caroline Dong, Lab Mentor

Introduction and Background

  • Heat stress is a major stressor in livestock production systems throughout the world [1]
  • The most effected organ by heat stress is the gut [1]
  • The effects of heat stress can be studied using bean beetles
  • Heat stress causes reduced fitness and survival in bean beetles [2]
  • Bean beetles were used as a model organism because they have a short reproductive cycle and are low maintenance [3]
  • Bean beetles are easy to handle and manipulate stress [4]
  • Bean beetles are easy to extract because eggs develop within the bean [4]

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References

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[1]Patra, Amlan Kumar, and Indrajit Kar. Heat Stress on Microbiota Composition, Barrier Integrity, and Nutrient Transport in Gut, Production Performance, and Its Amelioration in Farm Animals, 31 Mar. 2021, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071753/. 

[3]Hebert, Sadie. Authentic Research with Bean Beetles, University of Minnesota, https://cbs.umn.edu/hhmi-grants/projects/nonmajor/beanbeetles. 

[2] Ivimey-Cook, Edward, et al. “Inbreeding Reduces Fitness of Seed Beetles under Thermal Stress.” BioRxiv, Cold Spring Harbor Laboratory, 1 Jan. 2021, https://www.biorxiv.org/content/10.1101/2021.05.05.442709v1.full. 

[4] Beck, Christopher W, et al. “Effects of Evolutionary History on Adaptation in Bean Beetles, a Model System for Inquiry-Based Laboratories - Evolution: Education and Outreach.” BioMed Central, Springer US, 26 Mar. 2013, https://evolution-outreach.biomedcentral.com/articles/10.1186/1936-6434-6-5. 

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