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Mistakes and Lessons Learned Part 5: Maintaining Homozygous Cultures/Male Identification

View from the Bench / By Dan Richards and LeAnn Bowers

Summary:

In this five part series we are exploring some common mishaps when working with C. elegans, so you can hopefully avoid them too! Especially since when testing a specific variable (compound, strain, etc.) we want to limit all other variables that may skew results or lead to inaccurate data. In the final installation of this five part series, we ask: is it a problem that I identified several males while prepping for an experiment? What should I do about it?

Males exist in low frequencies under ideal laboratory conditions (≤0.002 for Bristol N2) and although variation exists between Caenorhabditis strains, it is always below 0.5. Identifying males in your C.elegans culture may be an identifier that the population has experienced some degree of stress, or the population has been grown for too many generations that has allowed outcrossing. 

For most experiments, 1-2 males per 1000 hermaphrodites will not heavily skew data and therefore can be picked off of plates once identified. Lifespan experiments are one of the assays that are more sensitive to the presence of males, because the lifespan of a hermaphrodite that has been mated by a male is significantly less than a self-fertilized hermaphrodite. Males are also smaller than hermaphrodites, therefore they should also be removed from experiments where the size of C.elegans is being taken into consideration for phenotype determination. In addition to the smaller size of males, they may also be identified by their behavior. Males will move alongside a hermaphrodite for mating. 

identifying male worms

A video identifying a smaller, male C.elegans.

References  

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  2. Yin, D., & Haag, E. S. (2019). Evolution of sex ratio through Gene Loss. Proceedings of the National Academy of Sciences, 116(26), 12919-12924. https://doi.org/10.1073/pnas.1903925116 
  3. Meneely, P. M., Dahlberg, C. L., & Rose, J. K. (2019). Working with worms:caenorhabditis elegans as a model organism. Current Protocols Essential Laboratory Techniques, 19(1). https://doi.org/10.1002/cpet.35 
  4. Stuhr, N.L., Curran, S.P. Bacterial diets differentially alter lifespan and healthspan trajectories in C. elegans. Commun Biol 3, 653 (2020). https://doi.org/10.1038/s42003-020-01379-1
  5. Kirienko, N. V., Kirienko, D. R., Larkins-Ford, J., Wählby, C., Ruvkun, G., & Ausubel, F. M. (2013). Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death. Cell host & microbe, 13(4), 406-416. https://doi.org/10.1016/j.chom.2013.03.003
  6. Eisenmann, D. M., Wnt signaling (June 25, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.7.1, http://www.wormbook.org.
  7. Wang, H., Zhao, Y., & Zhang, Z. (2019). Age-dependent effects of floxuridine (FUdR) on senescent pathology and mortality in the nematode Caenorhabditis elegans. Biochemical and Biophysical Research Communications. doi:10.1016/j.bbrc.2018.12.161
  8. Lucanic, M., Plummer, W., Chen, E. et al. Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects. Nat Commun 8, 14256 (2017). https://doi.org/10.1038/ncomms14256

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