Insect vectors as a mechanism of divergence and

sympatric speciation in microbial communities Cameron A. Gable1*, Victor H. Quach1, Turner S. Baker1, Kelly M. Thomasson2, and Stephen R. Proulx2

1University of California, Santa Barbara: Department of Molecular, Cell and Developmental Biology 2University of California, Santa Barbara: Department of Ecology, EvoluNon, and Marine Biology

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Introduction

Abstract Results

Methods

Discussion

Literature Cited

Acknowledgements

The mechanisms of speciation and divergence are both interesting and allusive. In particular, the events leading to sympatric divergence and speciation are still unclear. Previous research has indicated that a cell of the yeast species Saccharomyces cerevisiae is more likely to survive stress elements if it has already differentiated into its sporulated state. The fruit f ly, Drosophila Melanogaster, acts as one of these stressors by frequently ingesting these yeasts. Yeast cells with faster sporulation rates may therefore have a selective advantage when faced with stressors such as insects. In this study, we investigated the evolutionary effects of D. Melanogaster on the sporulation rate of S. cerevisiae. Using sporulation and competitive growth rate as comparative metrics, we exposed yeast cells to fruit flies, selecting only the survivors of ingestion for the following generation. Each generations phenotype was then compared to the first generation. After seven generations, our preliminary data indicate change in the sporulation rate from the first to the seventh generation, but this change is not yet significant. More expansive results from this study could shed light on the evolutionary effects insects on microbial communities and sympatric speciation events within these microbial species.

Diploid Mcherry strain M4330 was grown at 30 degrees Celsius in broth culture for 48 hours to initiate sporulation. The sample was then placed into two capillary tubes and put into a CaFe vial system containing 4 flies which had been starved for 12 hours. After 4 hours, the capillary tubes were removed and replaced with two capillary tubes, each containing YPD+tet+AMP+G418. After a 6 hour period of reenoculation, the flies were removed and the capillary tubes were expressed into YPD+tet+AMP+G418. The caf vial was also swished with YPD+tet+AMP+G418. Both of these samples were grown for 48 hours. The two samples were then mixed 1:1 and the process was then repeated with the new sample. The procedure was repeated for 5 generations. At the end of experimentation, growth rate and sporulation rate were measured and compared to original values.

Our greatest appreciations go to Dr. Stephen Proulx and Kelly Thomasson, who mentored us through the entire course of the project. We are also extremely grateful for the contribution of the UC Santa Barbara Undergraduate Research and Creative Activities (URCA) unit, as this project would not have been possible without their generous funding. Finally, we thank our Lab Manager, Simone Dupuey, for her valuable support and feedback throughout the completion of this project.

Figure 1: A and B Generational Growth Curves. Trials A and B are replicates of each other, both starting with a common generation 0 strain, Mcherry 4330. All generations were grown in YPD+tet+AMP+G418 liquid media. Optical density readings were taken using a plate reader at a wavelength of 600nm. The graph is plotted with time in seconds versus optical density at a wavelength of 600nm

Figure 2: Negative Control Generational Growth Curves. The negative control was grown from the same generation 0 strain as both replicates (see figure 1). All generations were grown in water at the same time replicates A and B were treated through the CaFe system. They were later grown in YPD+tet+AMP+G418 liquid media in between generations. Optical density readings were taken using a plate reader at a wavelength of 600nm

Optical densities are commonly taken over a period of time to assess microbial growth. As seen in figure 1, the growth curves of M4330 contains variation between each generation. In most instances, the logarithmic phase for each is steep and positive. This is analogous to a large increase in growth of the population and is commonly seen in microbial populations with sufficient food supplies and little suppressive factors. Three of the populations have peculiar log phases. One possibility this irregularity can be attributed to is the fly gut caused the populations to generate more diploid spores in order for survival, hence less cells for growth.

Figure 2 illustrates growth curves of each generation of the negative control. Peculiarly, three of the populations return to their original optical densities after a seemingly normal log phase and a large spike in growth. This can be credited to a shortage of nutritional content in the media due after very rapid growth. This may be an indication of a naturally faster-growing strain and therefore would support the conclusion that the fly gut stimulated sporulation of replicates A and B.

Figure 3 concludes that there was a significant difference between maximum growth rates of all generations. Multiple comparisons (Tukey) showed significant differences between generations 1 and 5, but not generations 0 and 5. Because of the small sample size, the growth rate of generation 1 can be seen as an irregularity, therefore, this does not support a change in either growth rate or sporulation rate.

Sporulation rate could not be explicitly measured as the strain Mcherry 4330 would not sporulate over the course of a week. This characteristic of high growth rate supports the data observed in figures 1 and 2 and still leaves room for the possibility that sporulation rate and other phenotypes can be experimentally provoked to change due to other controlled conditions.

The study of interspecies interactions such as that between D. Melanogaster and S. Cerevisiae supply vital information about community dynamics and the repercussions these interactions have on the ecosystem as a whole. In some cases, one species may have a strong enough effect on another to effectively select for a certain phenotype. Over time, this phenotype may attain a high enough relative fitness that the species diverges and sympatric speciation is observed.

The concept of competitive exclusion, or Gauses Law, states that two populations competing for the same resource cannot coexist when all other ecological factors remain constant [3]. Furthermore, when one population gains even the slightest competitive advantage, in the long term that population will flourish over the other. Fitness drives this exclusion as the population with higher fitness should out compete other populations [5]. On the other hand, coexistence theory states that if the ecological environment is evolving with a number of niches to occupy, it can in fact house multiple species despite their discrepancies in fitness values [3]. Interspecies interactions are commonly examined in a laboratory setting to analyze how these interactions may unfold in nature. Two model organisms in Drosophi la Melanogaster and Saccharomyces Cerevisiae commonly interact in the wild as the yeast serves as food to the fly [4,6]. One way to examine these two species in a laboratory is known as a caf system. This setup includes caf vials as the primary housing apparatus for the flies and capillary tubes containing the yeast being fed to the flies. This studys purpose is to examine the competitive ability of yeast populations in the strain M4330 when posed with a predator, D. Melanogaster. According to Coluccio et al. 2008, if the yeast cell is in a sporulated state when preyed on, it has a much higher potential to survive the fly gut than it would in a vegetative state [4,6]. This is due to the unique chitosan and dityrosine components of the spore ascopore wall [1,2]. Because of this, it is believed that over the course of multiple generations the populations repeatedly ingested by D. melanogaster may evolve to show characteristics of a faster sporulating strain. Furthermore, these frequently ingested yeast populations may diverge from other populations within its species that are infrequently ingested by insects.

[1] Briza, P., A. Ellinger, G. Winkler, and M. Breitenbach. 1988. CHEMICAL-COMPOSITION OF THE YEAST ASCOSPORE WALL - THE 2ND OUTER LAYER CONSISTS OF CHITOSAN. Journal of Biological Chemistry 263:11569-11574. [2] Briza, P., G. Winkler, H. Kalchhauser, and M. Breitenbach. 1986. DITYROSINE IS A PROMINENT COMPONENT OF THE YEAST ASCOSPORE WALL - A PROOF OF ITS STRUCTURE. Journal of Biological Chemistry 261:4288-4294. [3] Chesson, Peter. "General Theory of Competitive Coexistence in Spatially-Varying Environments." Theoretical Population Biology 58.3 (2000): 211-237. [4] Coluccio, A. E., R. K. Rodriguez, M. J. Kernan, and A. M. Neiman. 2008. The Yeast Spore Wall Enables Spores to Survive Passage through the Digestive Tract of Drosophila. Plos One 3:7. [5] Dejong, G. 1994. THE FITNESS OF FITNESS CONCEPTS AND THE DESCRIPTION OF NATURAL-SELECTION. Quarterly Review of Biology 69:3-29. [6] Goodman, A. L., N. P. McNulty, Y. Zhao, D. Leip, R. D. Mitra, C. A. Lozupone, R. Knight, and J. I. Gordon. 2009. Identifying Genetic Determinants Needed to Establish a Human Gut Symbiont in Its Habitat. Cell Host & Microbe 6:279-289.

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