1. Presented to the Pacific Ballast Water Group April 16 th, 2014 Brian Maurer Nick Welschmeyer Golden Bear Facility Moss Landing Marine Laboratories 1

2.  Introduction to Heterotrophs and Autotrophs in the 10-50 µm size class  Methods: Sampling and Flow Cytometric Analysis  Results  Conclusions 2

3. Cyclotella Autotroph (Diatom) Oxyrrhis marina Heterotroph (Dinoflagellate) Cannot synthesize its own foodCan synthesize its own food (Photosynthetic) 3

4.  Abundance and activity of autotrophs is easier to measure due to Chlorophyll a (chl a) fluorescence Chlorophyll a fluorescence  Readily distinguished from non-biological particles for flow cytometric or epifluorescent counting  Growth can be detected based on increase in chl a fluorescence over time (MPN)  Activity of chl a indicates “health” of autotrophic cells (PAM fluorometry)  Photosynthetic activity can be measured ( 14 C incorporation) 4

5.  What is the relative abundances of heterotrophs and autotrophs in the 10-50 µm size class? Protoperidinium sp. (heterotrophic dinoflagellate) feeding on a diatom chain (autotrophic) 5

6.  Live cells enumerated using Accuri C6 Flow Cytometer from December 2010-Janurary 2014 at a variety of locations, primarily along the West Coast, USA  Treated and untreated samples inoculated with Fluorescein diacetate (10µM) for detection of living cells  Files re-analyzed using FloJo© software for the following parameters:  Live autotrophs 10-50 µm  Live heterotrophs 10-50 µm  Live autotrophs 2-10 µm  Live heterotrophs 2-10 µm 6

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9. Forward Scatter (size) Red Fluorescence (Chl a)  Autotrophs distinguished from heterotrophs based on presence of chl a (red) fluorescence  Larger cells must have higher fluorescence to be considered autotrophic  Autotrophs make up <1% of total counts, bulk of counts are non-biological  Red excitation, red emission combination used to avoid fluorescein spillover 9

10.  Accuri C6 Flow Cyometer has 2 lasers: Blue and Red 10

11. 490nm513nm Source of fluorescence spillover into red detector Emission λ > Excitation λ 11

12. 430nm662nm 671nm Blue laser 488nm Red laser 633nm Red laser ensures no spillover of fluorescein fluorescence into red detector 12

13. Forward Scatter (size) Red Fluorescence (Chl a)  Size distinction based on forward scatter signal of bead standards  2.3 µm = 1.5x10 5 FSC  10 µm = 1.0x10 6 FSC  50 µm = 1.0x10 7 FSC  Majority of particles are <10µm 13

14. Forward Scatter (size) Red Fluorescence (Chl a)  Autotrophs 2-10 µm  Autotrophs 10-50 µm  Heterotrophs 2-10 µm (contains non- biological particles)  Heterotrophs 10-50 µm (contains non- biological particles) 14

15. Autotrophs 10-50 µm  Display population of interest (Autotrophs 10-50 µm) on plot of Size vs. Green (fluorescein) Fluorescence  Determine the count of cells which meet the “live” threshold based on intracellular accumulation of fluorescein fluorescence (indicates enzyme activity) Live threshold Forward Scatter (size) Green Fluorescence (Fluorescein) 15

16. Heterotrophs 10-50 µm  Display Heterotrophs 10-50 µm on plot of Size vs. Green (fluorescein) Fluorescence  Determine the count of cells which meet the “live” threshold  Less than 1% of “heterotroph” events in 10-50µm range are live cells Live threshold Forward Scatter (size) Green Fluorescence (Fluorescein) 16

17. Autotrophs 2-10 µm  Display Autotrophs 2- 10 µm on plot of Size vs. Green (fluorescein) Fluorescence  Determine the count of cells which meet the “live” threshold Live threshold Forward Scatter (size) Green Fluorescence (Fluorescein) 17

18. Heterotrophs 2-10 µm  Display Heterotrophs 2- 10 µm on plot of Size vs. Green (fluorescein) Fluorescence  Determine the count of cells which meet the “live” threshold  Approximately 0.1% of “heterotroph” events in 2-10µm range are live cells  Live threshold Forward Scatter (size) Green Fluorescence (Fluorescein) 18

19. n=113n=79 19

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27.  Majority (>80%) of live cell numeric counts for the 10- 50µm size class are autotrophic  Ballast water treatment causes no significant change in the relative proportion of heterotrophs and autotrophs  A higher proportion of heterotrophs exists in the smaller size class (2-10µm) relative to the 10-50µm size class  No strong seasonality effect was observed at most locations for the 10-50µm size class  Autotroph specific techniques will detect the majority of living organisms in the 10-50µm size class:  This may be sufficient for compliance monitoring  Effort should be made to quantify living heterotrophic organisms for Type Approval testing 27

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