1. Symbiotic Complexity: Discovery of a fifth Symbiont in the Attine Ant-microbe Symbiosis Authors: Ainslie Little & Cameron Currie Presented by Nikki Donathan

2. Define Fungal Symbiotic Associations  Leaf cutter ants share a mutualism relationship with the fungus they harvest  The term "mutualism" describes any relationship between individuals of different species where both individuals derive a benefit. http://myrmecos.files.wordpress.com/2009/06/ versicolor31.jpg

3. A Fifth Symbiont Factor  The success of the mutualism is threatened by specialized fungal parasites (Escovopsis)  Ants have a second mutualism with bacteria (Pseudonocardia) which produce antibiotics that inhibit garden parasite  Presence of fourth microbial symbiont associated: Black Yeasts (Ascomycota; Phialophora)

4. Black Yeast  Found in soil, plants, water, and decaying wood  Act as secondary saprophytes and oligotrophs  Cause disease in humans and plants http://www.clinicalfungi.org/i mages/atlas_kaft.jpg

5. Black Yeast Information  Black yeasts grow on the ant’s cuticle where the mutualistic bacteria is cultivated

6. Purpose of Study: Discovery of a fifth symbiont 1.Establish that black yeasts are symbionts of fungus-growing ants 2.Find that it grows consistently on the ant’s cuticle 3.Discuss the importance of additional layers of complexity in symbioses

7. Research Set-up  Black yeasts isolated and amplified from study were from colonies of fungus- growing ants collected between 2002 and 2006 in Panama, Ecuador, and Peru

8. Part 1: Isolation of Microbes  To isolate, the ants were scrapped with a sterile metal utensil and then the material was spread on chitin agar and incubated at room temperature for four weeks.  The colonies grown were then transferred to a potato dextrose agar

9. Part 2: Localization of black yeast  Isolation of different body parts of the ant workers  Head, thorax, and abdomen  Each section was placed in 500 ul of sterile distilled H2O, centrifuged for 30 seconds, and then 100 ul were spread on chitin agar  Thorax was further examined by scraping the dorsal, ventral, and lateral surfaces and plating them separately

10. Method continued  DNA was extracted from cultures collected and directly from the ants

11. Results  Part 1: Prevalence and Distribution of the Black yeast  Sampling of ant population in Panama canal zone indicated high prevalence of black yeast among Apterpsigma pilosum colonies  Also isolated from colonies in other regions of Panama, Peru, Ecuador

12. Results Continued  Part 2: Localization on Black Yeast on Ant  Most concentrated on thorax

13. Results continued  Black yeast frequently cultured from Apterostigma worker  Other attempts to isolate from other genera of fungus-growing ants were unsuccessful

14. Discussion  What we now know  Indicate that black yeast is a fifth symbiont  Present in every population of Apterostigma  Black yeast concentratted on specialized locations on ants’ cuticle where bacterial mutualists are maintained  Suggests that black yeast may have been present in early stages of evolution of complex symbiosis of ants and fungus

15. Further Research?  Suggest four scenarios regarding evolutionary origin of black yeast: 1.Ants acquired by manuring their gardens with vegetation containing the endophytes 2.Black Yeast associated with ancestral ants of fungus growers 3.Black yeast underwent a host shift (host insect to ant) 4.A Black yeast- Pseudonocardia grow together in some soils

16. Further Research continued  Suggest further studies to tease apart interactions between black yeast and anti-microbe symbiosis  Exploration of black yeast role may indicate antagonizes the ants’ mutualistic bacteria

17. Questions?

18. Reference  Ahmadjian, Vernon & Paracer, Surindar. Symbiosis: An Introduction to Biological Associations. “Fungal Associations of Protozoa and Animals.” P 101-102. Oxford University Press. 2002.  Currie, Cameron R & Little, Ainslie E. F. “Symbiotic Complexity: Discovery of a Fifth Symbiont in the Attine Ant-microbe Symbiosis.” Evolutionary Biology. 2 July 2007. Accessed 23 September 2009. http://web.ebscohost.com/ehost/detail?vid=1&hid=12&sid=0f2d9d 3e-0c87-406b-8c1f- 52fa502e5894%40sessionmgr11&bdata=JnNpdGU9ZWhvc3QtbG l2ZSZzY29wZT1zaXRl#db=aph&AN=4647127