1. Using Microbial Ecology to Teach Experimental Design and Sampling Methods Mary E. Allen, Hartwick College Ruth A. Gyure, Western CT State University American Society for Microbiology MicrobeLibrary POWERPOINT SLIDES TO SUPPLEMENT EXERCISE 2: Microbial Community Structure and Organization SEM of Bacteria in Mouse Ileum. Source: Ken Rozee et al., Microbe Library, ASM

2. PART I Introductory material that will help instructor prepare and execute the exercise

3. REVIEW CONSIDERATIONS WHEN SAMPLING MICROBES IN THE NATURAL ENVIRONMENT: 1. Detection and visualization 2. Definition and differentiation of taxa 3. Cultivation issues and ecological relevance 4. Interdependence (consortia) 5. Adherence (biofilms)

4. Current, genetic view of species-level difference: DNA-DNA hybridization rate of about 70% 16s rDNA similarity of >98% Figures from Ogunseitan, Microbial Diversity WHAT IS A PROKARYOTIC SPECIES???

5. These isolates are all in the same genus. Why so different????? THEY CHANGE MORPHOLOGY DEPENDING ON THE CULTURE MEDIUM!

6. CAN I ISOLATE? IS THE ISOLATE ECOLOGICALY RELEVANT? Photo credit: D. J. Patterson, L. Amaral-Zettler and V. Edgcomb. Courtesy of micro*scope. There is a wide diversity of organisms in the marine sample illustrated above. However, when plated on standard growth media in the lab, very few of these species will grow. Even if an isolate is obtained, how do we know it is one that plays an important role in the system?

7. This pictures was made using "Fluorescent in Situ Hybridization" (FISH). The organisms in red represent the anaerobic methanotrophic Archaea (ANME) and the green organisms are sulfate reducing bacteria (SRB). Picture made by C. Jagersma at the Max Planck Institute Bremen. MICROBIAL CONSORTIA Example: AMETHOX See notes that accompany this image.

8. BIOFILMS S. Lowry—University of Ulster—Stone/Getty Images Elude traditional sampling and visualization methods Cells tightly clumped and difficult to separate, isolate, identify Phenotypic variation difficult to assess after sampling

9. Diversity: Species composition, i.e. how many different species are there, and how are the numbers distributed among them Structure: How are these individuals distributed and organized in the environment?

10. These simplistic diagrams illustrate some possible ways in which organisms may be distributed in the environment. Actual distribution will show combinations of these patterns, and will also differ depending upon the scale at which you observe, sample and and measure it.

11. Terrestrial environment: Soil Crumb Photo and Diagram from Brock Biology of Microorganisms, Madigan and Martinko MARINE “SNOW” ‘Hot spots’ of bacterial concentration and activity, contributing to global cycling of carbon and nutrients Azam and Long, Nature, 2001 These examples illustrate that microbes will organize themselves in response to environmental conditions and interactions at very small scale; However, such interactions may not be of interest when larger scale systems are being studied

13. SCALING – At what scale does one need to sample, observe, and measure in order to answer the question posed? Pictured here – from global scale (oceans) to regional and local scales; intercellular scale (symbiont community in a protozoan population)

14. STATISTICAL METHODS AND PRIMER, ENVIRONMENTAL APPLICATIONS: http://epa.gov/bioindicators/statprimer/ BRAINSTORM: HOW DOES ONE MAKE DECISIONS ABOUT SAMPLING? 1.Size of population, community or system, area of interest 2.Scope of study? 3.Budget? 4.Variability (standard deviation, error)? depends on heterogeneity, abundance, distribution, both spatial and temporal, method, etc. (See link below for great discussion about statistical considerations ) 5.Technological ability (Can one directly observe organisms or cells? Can one target individuals or groups with specificity? How precise are the units of measurement?) 6.Experimental approach 7.SCALE! (many issues)

15. Sampling soil microbes in a relatively static soil community Even here – there are challenges of scaling, and dealing with localized heterogeneity that could mask larger scale changes of interest.

16. EXERCISE 2: Let’s “sample” some diagrammatic representations of microbial communities! In this exercise, sampling and measuring diversity alone is not the goal. We would also like to sample in a way that informs us about the distribution of organisms present.

17. HERE IS AN EXAMPLE OF ONE COMMUNITY THAT MAY BE ASSIGNED

18. BEFORE YOU RECEIVE A COMMUNITY, YOU MUST DECIDE ON A PLOT SAMPLING SCHEME. 1. STEP 1: Trace your 3 large plots onto the transparency after deciding on a placement plan. When finished, ask instructor for your sample community. 2.STEP 2: Record the number of individuals for each species observed in each plot on your data sheet. 3.STEP 3: Discuss the following questions as a group, as a class – or later as a homework assignment.

19. a.How is this community different from one that might exist in the water column of a lake, for example? b.If you were to imagine a habitat that this simulated diagram might represent, what would it be? c.When sampling a community of this sort, does the scale of the area of investigation matter? In what ways will it affect your sampling approach? d.By taking data from 9 small plots instead of the 3 relatively large plots you just did (adding up to same comparable area) – which do you feel would give a more realistic or accurate picture of the actual community? What are the advantages and disadvantages of each approach? Do you think each method would give the same calculated Simpson’s index? Why or why not? e. Finally, would a line transect approach be appropriate for sampling this community? Why or why not?

20. STEP 4: Repeat the sampling this time tracing 9 small plots rather than 3 larger ones. You must use same rules for the placement scheme! Record data as before STEP 5: Draw 3 transect lines and and determine a placement scheme. Record data. STEP 6: Calculate Simpson’s index of diversity for each of the 3 sampling approaches.

21. PART II Post-exercise discussion and review In the next 3 slides you will see what the 4 communities actually looked like, and an example of how the 3 sampling schemes might be applied to Communities C and D (with a sample data set for class discussion)

22. D A C B

23. v v C D

24. EXAMPLE OF HYPOTHETICAL DATA SiteNumber if individuals (count) C Small plots Large plots Transects Species123456789total123 123 A0003001004.00000.0000 B0005000005.00000.0000 C0001000001.00000.0000 D0001000001.00000.0000 E 8008.00000.0 F 180018.00000.0 G 2002.00000.0 H 1001.00000.0 I J K L M 11.029.00.0 SiteNumber if individuals (count) D Small plots Large plots Transects Species123456789total123 123 A8000001009.065819.01102.0 B3210001029.056415.02103.0 C2211102009.041611.00000.0 D1300001005.0131 0101.0 E1400000106.01225.00000.0 F116010000523.01291031.01001.0 G21600001010.0424 3014.0 H01402040011.01001.02024.0 I0050020007.01001.05117.0 J0000010001.0001 010 K0000020002.075820.00202.0 L005014000019.089623.01023.0 M0031110000125.03126.00033.0 136.0148.031.0

25. Some examples from the literature to review and discuss concepts of diversity, scaling, distribution

26. Drop-size soda lakes ( Qvit-Raz, Genetics, 2008) This is an amzing study showing actual differences in complex communities that go through successional stages in tiny drops that form from dew and exudates of the Tamarax tree which releases salty compounds. An ecosystem where large diversity at very small-scale was observed, relating to microgradients of physical and chemical variables.

27. Ley et al., 2006 GUT MICROBIAL COMMUNITY Is a community with a large number of closely related species more diverse than one with fewer numbers of more distantly-related ones? Here we notice that although the gut community is known to be “very diverse,” microbial mats in nature tend to show far greater diversity when taxa are differentiated at higher group levels rather than species level. Our view or estimate of diversity will depend upon how we decide to define it…

28. BIOGEOGRAPHY: IS EVERYTHING EVERYWHERE??? Fenchel and Findlay’s work, as reported in Science, 2005 See notes that accompany this slide

29. PNAS, 2007 What types of environments on earth are “most diverse?” Why do we care? (This study suggests that generally speaking, at a global level, salty environments are more diverse than freshwater or soil)

30. Findlay 1982: Both approaches yield similar estimates of abundance, but larger scale sampling tends to give poor representation of distribution/patchiness

31. WHAT ABOUT MOLECULAR ANAYSIS OF MICROBIAL COMMUNITIES? Ranjard et al. 2003 tested effect of SIZE of soil sample used in molecular community analysis. For bacteria, SIZE OF SAMPLE did not affect ability to distinguish unique communities from one another. For fungi, samples <1g may not be adequate. HOWEVER, in larger samples, technique of DNA extraction is biased toward dominant organisms and may not accurately assess diversity. FOR BACTERIA: Many subsamples are often taken, and it is the selection of the sampling location that will give best indicator of larger scale abundance and diversity

32. Desmarais 2002 When is use of transects appropriate? One example: when one hypothesizes a gradient relationship and wishes to test it using regression analysis, see below! In this study, researchers looked for change in numbers of fecal indicator organisms across various transects in relation to water’s edge. Keep in mind, most aquatic depth sampling is, in effect – transect sampling

33. FINAL THOUGHTS: The question being asked drives the experimental design. The practical limitations of sampling limit the type of question that can be asked – and answered! Pilot sampling is always essential to assess the nature of the system!! Students will better understand scientific process when we appreciate the considerations that go into every scientist’s sampling and measurement plan!