Jason E. Adolf and Judy K. Walker UHH Marine Science Flow Cytometric Analyses and RTCM of Water Quality around Hawaii Island Jason E. Adolf and Judy K. Walker UHH Marine Science

Study sites Hilo Bay Pauoa Bay Hilo Bay (E. Hawaii) Pauoa Bay Hawai‘i Island Pauoa Bay Hilo Bay (E. Hawaii) Rivers Pauoa Bay (W. Hawaii) SGD SGD 1 km 100 m Leeward side Low rainfall Ground water plume Windward side High rainfall Groundwater plume(s)

EPSCoR MARINE sites, including Pauoa Bay Marine ecosystem response to environmental variation, such as climate change (atmospheric-oceanic) and submarine groundwater discharge Pauoa Bay Hawai‘i Island Kīholo Plot The Marine Agenda is developing research capacity to investigate ecosystem responses to primarily environmental factors, such as climate change, ocean warming, and groundwater discharge. The research activities will be conducted in the two marine plots established on the Kona coast of the Island of Hawaii in the watersheds where terrestrial plots were established. Kīholo Bay Kaloko Plot 3

Why examine phytoplankton responses to coastal hydrology? A complex, fast-responding microbial assemblage many types, different needs, different fates A good ‘indicator’ Biomass serves as a nutrient vector (down); productivity; HABs Size structure responds to environmental forcing Size structure can determine the ‘fate’ of phytoplankton -sinking rate -grazing losses Environment Phytoplankton Ecosystem

Flow Cytometry What can a flow cytometer do? Flow Cytometry is an important tool for our research What can a flow cytometer do? Count cells (up to 10,000 per second) Optically classify each cell, one at a time, 10,000 per second Can distinguish phytoplankton from other cells Quantify optical properties of cells and generate statistics Side and forward scatter – indicator of cell size and shape Fluorescence (green, orange, red) – indicator of pigmentation

Flow Cytometry Optics Laser Dichroic Filters Flow cell Bandpass PMT 4 Dichroic PMT Light scatter detectors Filters 3 Flow cell PMT 2 Bandpass Filters PMT 1 Laser J. Paul Robinson, Purdue University

Calibration with size standards (latex beads) FSC-H and FSC-A poorly resolved 1 & 2 micron beads SSC-H and SSC-A didn’t have this problem The machine needs to be calibrated against phytoplankton for absolute sizing

Flow Cytometry: a monitoring tool for Hawaiian waters Exploring two parameters from each sample Total phytoplankton (cells / mL) Sum events within regions ‘Avg ESD’ Weighted average of cell diameter F = fractional count for the region ESD = geo. mean ESD for that region Avg ESD = (F1*ESD1)+ (F2*ESD2)+ (F3*ESD3)+ (F4*ESD4) Red Fluorescence Bacteria are analyzed similarly in each sample by applying a fluorescent stain Cell Size

Phytoplankton in SGD plumes of West Hawaii Island Counting and optical characterization Abundance, size and pigmentation Groundwater-influenced waters Typical off-shore (surface) Kaloko: near-shore (surface) Kiholo: near-shore (surface) Chlorophyll fluorescence Size

Larger phytoplankton are constrained to surface plume (Kaloko Bay) Depth profile of salinity and phytoplankton (determined by flow cytometry) from Station 14 on July 27, 2010. Middle FSC-A = forward scatter area (size) FL3A = levels of red fluorescence Bottom

Euglenoid blooms of West Hawaii (green / black water) A Euglenoid causes the dark water -Not toxic It’s a large cell with potential to sediment, but the blooms are localized Reduced circulation set favorable conditions for the bloom to occur We now have a tool for monitoring this Euglenoid to learn better how it fits in to the ecosystem Euglena-like cells ~70 mm Synechococcus

Phytoplankton Characteristics at Different Sites of West Hawaii Euglenoid bloom Kīholo Plot Kaloko Plot Pauoa Bay

Conclusion Research – Do phytoplankton ‘respond’ to nutrients in SGD? Approach: Flow cytometry A tool to address this question (numbers and size of phytoplankton) Developing a broadly applicable monitoring tool

Real-time Continuous Water Quality Buoy Deployed in Hilo Bay Western portion of Hilo Harbor Moored with >100 lbs steel Want to capture storm / base flow conditions April 7, 2010 Still there! Tides FW SGD Buoy for Kiholo Bay is ready to go!

Hilo Bay WQ Buoy USCG beacon Data logger / cell phone modem bumper Solar powered ~6 feet float waterline probes ballast / battery Mooring attachment YSI EMM 68

Hilo Bay Buoy Data: Salinity and Wailuku Flow

Apx one day lag between buoy and stream gauge Peak discharge Minimum salinity

Storm Surges: Turbidity in Hilo Bay

Turbidity subsides before salinity ‘recovers’ Nov 7, 2010 – Hilo Bay From Rt 19 overlook Turbidity subsides before salinity ‘recovers’ Hamakua coast

Hilo Bay Chl a and Salinity

Storms flush Chl a from Hilo Bay Recovery of Chl a? Vertical migrations?

Conclusions Many tools in place at UH Hilo to examine hydrologic influences on coastal phytoplankton / microbes Developing indicator tools Understanding the influence of groundwater and surface water is a key research drive Linkages to terrestrial biomes is key

Acknowledgements NSF EPSCoR III (UH System) Tracy Wiegner (UHH Marine Science) Students: Judy Walker, Erick Johnson, Rebecca Most, Gillian Wysock, Ambyr Mokiao-Lee, Javez Mooteb, John Burns (Corals) NSF EPSCoR III (UH System) Kiholo / Kaloko Fairmont Orchid Green Committee Pauoa Bay NSF EPSCoR II (UH System) Hilo Bay

Conclusions East Hawaii – Ground water nutrients likely fuel productivity; Storm flows flush Hilo Bay, reduce productivity. How has the biology adapted to these conditions? West Hawaii – Phytoplankton differ between SGD / non-SGD plume areas Physical conditions limit use of nutrients