The significance and future potential of using Microbes for assessing ecosystem health: The Great Lakes example

1. The significance and future potential of using Microbes for assessing ecosystem health: The Great Lakes example Lightmicroscopy of Chlamydomonas sp.

2. Historically, chemistry and general limnology have been the major focus of research in the Great Lakes, with little emphasis given to their biota and their ecology. (Vollenweider quote) • …becoming increasingly apparent that it is essential to undertake such ecosystemic, integrated and holistic assessments of the food web structure and its various components from bacteria to fish….

3. Phytoplankton bloom around Shetland Isles Courtesy of Earth Observatory

4. …focus on the abundance, role and significance of the Great Lakes microbes and their potential role in assessing health of large ecosystems. Planktonic terminology: microplankton, 20-200 microns dia. nanoplankton, 2-20 microns picoplankton, 0.2-2 microns Thalassionema (or Thalassiothrix?) Courtesy of Bigelow Lab.

5. Phytoplankton analyses have been carried out since the end of the last century (1900) Fisheries and Oceans Canada embarked on extensive lakewide surveys since 1969. Size fractionation techniques, pioneered by author enabled classification by size. Epifluorescence microscopy

6. Above: UV light, individual bacteria and flagellates visible by DAPI-induced blue fluorescence. Below: blue light , Yellow and smaller red fluorescing picoplankton cells visible. Only the autofluorescence of the natural photosynthetic pigments can be seen. The large, very bright cell is a dinoflagellate, about 20µm).

7. Extensive studies on Great Lakes in late summer 1988 and 1989. Average bacterial abundance: 0.7 to 2.2 million per ml. In contaminated waters: 0.7 to 1.2 million per ml. At end of municipal waste disposal pipe: 3.4 million per ml. Positive relationship found between trophic status and microbial biomass

8. Autotrophic picoplankton (APP), heterotrophic nanoflagellate (HNF) and bacterial cell numbers were low in the oligotrophic areas, North Channel, Georgian Bay and main Lake Huron and considerably higher in the meso-eutrophic to eutrophic lakes Michigan, Erie and Ontario. HNF of unknown importance, abundance 400 to 3000 cells per ml. Choanoflagellates Courtesy of Bigelow Lab.

9. Population dynamics are poorly researched at time of writing. Ciliates are, however considered the top predators in the ‘microbial loop’ Two hypotrich ciliates: Euplotes (left) and Stylonychia (right)

10. Feeding of ciliates has great impact on food web structure. They provide a link to the larger crustacean zooplankton, transferring the microbial production to higher trophic levels. An earlier survey found abundance to be between 2 and 10 cells per ml, with 28 cells per ml in contaminated areas. So, what is the impact of nutrient enrichment and other stresses?

11. Bioassays were carried out in the lab, after developing rapid collection and concentration techniques. Metals were tested and the level considered ‘safe’ for the biota caused “overwhelming inhibition of carbon assimilation”. Research also included monitoring the components of the food web at various levels to evaluate impact on structure and function and to test the usefulness of microbes as early warning systems

12. Great Lakes Areas of Concern: 43 areas that needed immediate attention were identified and measures implemented for their decontamination. Data collected as part of this program compared picoplankton in contaminated and relatively clean areas. ANOVA tests showed that APP were significantly lower in comtaminated areas but there was no SSD where the bacteria and HNF were concerned. This indicates APP sensitivity to stress

13. Microbial community appears to have potential as an early warning and rapid bioassessment tool. Application also in sediment assays. Assay of contaminated sediment from Toledo Harbour suggested that the toxicty resulting in a reduction of picoplankton might have been caused by dissolved forms of zinc and manganese

14. Cytology. Picoplankton being discovered and named. Use of transmission electron microscopy proves two principal groups, eukaryotes and prokaryotes. TEM is invaluable for identification of both healthy and unhealthy cells thus providing a measure of health status. TEM can be used where cells are too small for epiflourescence microscopy.

16. Freshwater Autotrophic Picoplankton Review C. Callieri and J.G. Stockner Journal of Limnology 2002 Courtesy of Liverpool University, UK

17. Picoplankton Autotrophic Heterotrophic Prokaryote Eukaryote Also size classification: Micro 20 - 200 μm Nano 2 - 64 μm Ultranano <2 μm Ultra <5 μm Pico 0.2 - 2 μm Femto 0.02 - 0.2 μm

18. 1911 Lohmann called small plankton ‘nanoplankton’. 1955 Rodhe described μ-algae in Swedish Lakes. Algae in this size range also known as LRGT (little round green things). 1956 Experiment showed much photosynthesis achieved by organisms <64μm 1970s and 1980s Epifluorescence microscopy Flow citometry Electron microscopy Immunofluorescence techniques Chromatographic techniques Photo by Peter Parks. Courtesy of Image Quest.

19. Study Methods Preservation of samples Fixing Counting Size fractionation Time element Genetic differentiation Calculations • A member of the • Bacillariophyceae Courtesy of Clemson University

20. For Elise and Lord Kelvin Calculation of primary productivity at each depth

21. Species composition and diversity Still working on it! So far, three genera of single-cell picocyanobacteria, more colonial and very little knowledge of any crossovers. Most common eukaryotic APP is Chlorella (LRGT). Due to the size classification this can include many that could be nanoplankton

22. Ecology Found in a variety of freshwater systems, including ultra-oligotrophic lakes like Lake Baikal, high altitude, polar and subpolar lakes, and shallow eutrophic lakes or ponds. There is a pronounced difference in APP species composition along trophic gradients and seasonally within a lake. Single-cell picocyanobacteria are very abundant in oligo- to mesotrophic lakes, while colonial forms occur mostly in meso- to eutrophic lakes or ponds. Merismopedia tenuissima, a colonial cyanobacteria. Courtesy of Maryland Dept. of Natural Resources

23. Eukaryotic APP generally an order of magnitude less than picocyanobacteria. In temperate regions, tend to show peak abundance in spring or early summer during isothermal conditions of 5-10 °C. Eukaryotic cells dominate APP in acidic dystrophic and eutrophic lakes. Apparently, picoeukaryotes progressively replace picoprokaryotes in lakes with increasing nutrient concentrations and decreasing pH and they tend to be more abundant in the epilimnion in August and September, during periods of nutrient limitation. Depth profile of chlorophyll Courtesy of URI.

24. In Lago Maggiore the pico size fraction account for 35% of total plankton carbon annually. In many other water bodies the importance of pico fraction has been highlighted by indirect estimates. Thus, an UV-B inhibition of pico- plankton activities would likely affect the whole organic carbon cycle of the lake.

25. Paucity of data on seasonality of freshwater APP primary productivity ….. relative contribution of APP to total phytoplankton production rarely based on frequent samplings but average of the summer or occasional samplings. Highest percentages of APP productivity have been measured in Lake Baikal, where 80% of 14C uptake was in the <3 µm size fraction. In lakes of western Canada, the relative contribution of picoplankton to total photosynthesis ranged from 29 to 53%, in Lake Constance, Germany, relative contribution was 5-65%.

26. Processes of cell growth and cell division dependent on light and temperature in some cases, but on an endogenous circadian clock in others. Cell division reaching a maximum in the afternoon triggering an increase in the cell number, which then proceeds in the dark. APP loss comes from grazing, viral lysis, parasitism, aggregation and sedimentation. Heterotrophic nanoflagellates and small ciliates are the most important APP grazers. APP plays an important part in the microbial loop and is at the base of the carbon biomass food web.

27. Concluding Remarks Reliance on application of new molecular methods to study genetic diversity. Questions of how the APP will respond to changing climate, both UV-B radiation and temperature change. Lakes may be warmer and more strongly stratified with a severely nutrient depleted euphotic zone. What effect on the APP and the food chain? APP are the most ancient survivors of primordial seas and the primary carbon producer in aquatic ecosystems. Capable of adapting to extreme environments, they are distinct in many ways from all other algal groups

28. Example of environmental effects on LRGT The green alga Phacotus lenticularis - the type species of the chlamydophycean algal family Phacotaceae - is an unicellular flagellate of 13-17 µm in diameter. In a calcium poor medium In a calcium rich medium

29. From Jerry Evans’ website and mostly collected in Bell County, Texas. http://www.vvm.com/~jevans/ Botrycoccus

30. Pandorina Pleodorina Protozoans • Actinophrys Arcella Closterium Centropyxis Mystery ciliated protozoan

31. References Liverpool University : www.pcweb.liv.ac.uk/BAMBERI/Ugcourses/F901.htm Image Quest: http://www.imagequest3d.com Maryland Dept of Natural Resources: http://www.dnr.state.md.us/index.asp URI, University of Rhode Island: http://www.gso.uri.edu/criticalscales/about/kinds/phyto/phytotxt.html Clemson University: http://people.clemson.edu/~sadvs/index.html Begelow Laboratory:http://www.bigelow.org/inves.html