Microbial Grazers Lab

Transcription

1 Microbial Grazers Lab Objective: Measure the rate at which bacteria are consued by predators. Overview Size based food webs Microbial loop concepts acterial predators Methods to assess icrobial grazing rates Readings (see class web site) 1. Caron,D.A. et al. (2012) Marine Protistan Diversity, Annu. Rev. Mar. Sci. 4: Aza et al. (1983) Marine Ecology Progress Series, 10:

2 Size Classification (Revisited) Unlike terrestrial systes, priary production in aquatic systes is doinated by icroorganiss with sizes typically less than 200. Fetoplankton: Mostly viruses Picoplankton: acteria, cyanobacteria Nanoplankton: 2-20 Flagellates, dinoflagellates Microplankton Diatos, ciliates. Mesoplankton > 200 Zooplankton (copepods) Size classifications are used because: Functional definition (filter cutoffs) Feeding approxiately based on relative sizes Identification not always helpful acteria: (1 ) Typically 1-2 culture, or < 1 natural environents. Scheatic of size based feeding. Soe organiss ight be ixotrophs (both auto- and heterotrophy, shown in green), and soe ay feed across trophic levels (blue lines), or feed on organiss larger than theselves (red lines).

3 Nanoplankton Exaples (2-20 ) Flagellates Diatos

4 Microplankton Exaples ( ) Dinoflagellates Diatos Zooxanthellae, coral sybionts, are dinoflagellates. Their expulsion fro coral anial is what is called coral bleaching

5 Mesoplankton Exaples (> 200 ) Krill copepod

6 Phylogenetic Marine Protistan Diversity Mitochondrion Gra + Purple bacteria bacteria Chloroplast Cyanobacteria Flavobacteria acteria Green non-sulfur bacteria Theroproteus Methanococcus Pyrodictiu Archaea Multicellular Anials Extree halophiles Slie olds Fungi Methanobacteriu Entaoebae Plants Therococcus Theroplasa Eukarya Ciliates Flagellates Trichoonads Therotoga Aquifex Diploonads Microsporidia Fro Caron et al. (2012)

7 Classic Food Chain The classic view of aquatic food webs was the linear food chain fro phytoplankton to fish. Although bacteria were know to exist, they were not thought to be significant consuers of carbon or energy. icroplankton esoplankton CO 2 P Z F DIN P: Phytoplankton (e.g., Diatos) Z: Zooplankton (e.g., Copepods) F: Fish (both planktivors and piscivors)

8 acterial vs Phytoplankton Productivity Developent of epi-fluorescence reveals large nuber of bacteria (10 6 l -1 ) Developent of bacterial productivity assay shows large fraction of NPP is processed by bacteria (50%?). Fro: Cole et al. MEPS (1988). CO 2 P NPP P DOM? Copepods cannot eat bacteria though

9 Microbial Loop The icrobial loop is a conceptualization by which DOM can be routed into the classic food chain via bacteria and their grazers. (Poeroy 1974, Aza et al. 1983) Sources of C for food webs CO 2 P Z F nf Microbial Loop C DOM: Dissolved organic atter P: Phytoplankton Z: Zooplankton F: Fish : acteria nf: Nanoflagellates C: Ciliates

10 Is the Microbial Loop a Link or Sink for OC? Efficiency U R P U = P + R = P U Typical Efficiencies: How uch bacterial C akes it to zooplankton via the icrobial loop? CO P NPP Z A significant aount of research focuses on easuring growth efficiencies and feeding rates DOM 100 nf C Z nf nfc CZ 10% %

11 Top Down or otto Up Liitation? Energy otto up: Nutrient Liited P Priary producers Resources R Self organization C Top Down: Consuers Excessive Grazing Pressure Heat (Entropy) oth views are yopic, in that they are transient assessents and likely to change over tie, but the tie scale ay be long (decades or ore). Likewise, the icrobial loop, as a link, ay be iportant over short periods when food resources are scarce.

12 Protozoa Single-celled, eukaryotic, heterotrophs ranging in size fro 2 to 1 or ore. Feed ostly by phagocytosis (engulfent). Three basic types: Flagellates Use one or two (soeties ore) flagella (little whips) for otility. Size: Representative taxa: Dinoflagellates, Chrysoonads, icosoecids, Choanoflagellates, Kinetoplastids Ciliates Range fro uniforly covered with cilia (hair-like tubules) to ostly naked with tufts of cilia. Size: Representative taxa: (planktonic) Oligotrichs, Tintinnids, Scuticociliates, (benthic) Hypotrichs, Peritrichs, Heterotrichs Sarcodines Aoeba-like species without flagella or cilia. Many possess skeletal structures or shells Size: 5 to > 1 Representative taxa: Gynaoebae, Testacea, Forainifera, Radiolaria, Acantharia, Heliozoa

13 Feeding and Motility Phagocytosis Cilia used fro otility and particle feeding Flagella used for otility Extree exaple of phagocytosis (Schiegeli Acanthopagrus (youtube))

14 Heterotrophic Nanoflagellates (HNF) These are soe of the sallest eukaryotes (2-3 ) Typical HNF densities around l -1 Pictures fro

15 Ciliates Ciliates are protozoans (single cell) that can be identified by the cilia that surrounds ost of the body. Classic exaple is the Paraeciu sp. Also see: Densities around l -1 Two hypotrich ciliates: Euplotes (left) and Stylonychia (right) Colony of Carchesiu Paraeciu Tetrahyena

16 Ciliate Diversity (lakes and rivers).j. Finlay and G.F. Esteban (see

17 Dinoflagellates and Toxic bloos Dinoflagellates are the cause of red tides. Production of neurotoxins lead to fish kills and paralytic shellfish poisoning. See Harful algal bloos appear to be on the rise, due to eutrophication and global change? Noctiluca sp. Cyanobacteria

18 Techniques for easuring feeding rates Metabolic inhibitors Use an inhibitor (i.e., antibiotic) specific for eukaryotes. Measure increase in bacterial nubers in the presence and absence of inhibitor. Size fractionation Dilution ethod Radiolabeled bacteria Fluorescently label particles Filter predator out of saple, and easure bacterial growth. Measure bacterial growth rates at several saple dilutions Feed predators radiolabeled bacterial Feed predators fluorescently labeled particles or bacteria.

19 Metabolic inhibitors and size fractionation Size Fractionation Method Metabolic Inhibitor Method Unfiltered seawater Unfiltered seawater Eukaryote inhibitor 0.6 Filter d G dt A G A A G A Measure bacterial nuber increases in treatent A s copared to treatent s. Probles: Filtration can cause cell lysis. Inhibitors ay not be perfectly selective, and y be consued by bacteria. Cannot look at species-level grazing. Incubation tie is long.

20 Dilution Method Apparent Specific Growth Rate, A (d -1 ) 100% Unfiltered SW 80% Unfiltered SW Measure bacterial nubers at t-zero, and again at a later tie (one or ore days). Calculate apparent bacterial specific growth rate for each: A =ln(x(t)/x(0))/t Plot versus fraction unfiltered water. 60% Unfiltered SW 40% Unfiltered SW μ A = μ f Theoretical bacterial specific growth (no grazers), 20% Unfiltered SW Dilute with 0.2 filtered sea water 2 1 -Slope: grazing ortality, * Fraction Unfiltered Water (f ) Probles: Dilution alters syste. Cannot look at species-level grazing. Incubation tie is long.

21 Grazing Rate fro Dilution Cultures U G C R Mass balance around bacteria: f A * C G M M K if K K G K M A * fg G f G K M A * * * G K M Grazer uptake: * Mortality (1/d) f Fraction unfiltered * Denotes conditions in original saple G K G K G C R U dt d A M M * * * * G where, Line fro dilution plot: What is *? acteria production: R U Assuption behind dilution technique

22 Fluorescently or Radiolabeled bacteria or particles Water saples Added FLP or RLP at <20-50% of natural bacterial abundance At specific ties, take saple and preserver. Filter saple on 0.8 filter to reove unconsued particles. Either icroscopically count abundance of ingested particles per specific group of protozoa, or easure radioactivity. Accounting for bacterial abundance relative to added particles, calculate total nuber of bacteria consued per protozoan per unit tie. Can also calculate total bacterial reoval rate. Advantages: Can obtain species specific grazing rates Short incubation ties. Probles: Predators ay discriinate against particles.