Who is there - and what are they doing? A case study with harmful algae

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Who is there - and what are they doing? A case
study with harmful algae

• Sonya Dyhrman, Biology Department
• Woods Hole Oceanographic Institution
• EARTH Workshop - July 2008

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Microbial oceanography

• Marine microbesare beneficial and important!

• produce and consume green house gases
• recycle organic matter
• power the marine food web
• account for roughly half of global primary
  production (every other breath you take!)

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Outline

• Introduction to Microbial Oceanography
• What are Harmful Algal Blooms?
• Who is out there?
• Detecting Alexandrium catenella in the Puget
  Sound
• What are they doing?
• Detecting alkaline phosphatase activity

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The science you teach inspires!
2003 Publication in Limnology and
Oceanography Urease activity in cultures and
field populations of Alexandrium (previously
Gonyaulax)
1989 Bellarmine High School Marine Chemistry
Project Analysis of Parameters Affecting
Gonyaulax catenella Concentrations in
Quartermaster Harbor, Washington
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1989
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1989
2003
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1989
2003
Thank you Mr. Nilsen!
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Outline

• Introduction to Microbial Oceanography
• What are Harmful Algal Blooms?
• Who is out there?
• Detecting Alexandrium catenella in the Puget
  Sound
• What are they doing?
• Detecting alkaline phosphatase activity

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What are harmful algal blooms?

• Proliferation or bloom of a marine
  algae(phytoplankton)
• Common name is Red Tide
• Not all blooms are red
• Toxic events can occur without water
  discolorations
• Toxic impacts can occur at low cell number

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Two modes of harmful impacts from blooms

• 1 Non-toxic effects, typically from high biomass
• Reduced light
• Oxygen depletion (hypoxia/anoxia)

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Brown tide

• Aureococcus anophagefferens
• Small cell from the Pelagophyte group of algae
• Non-motile
• No known toxin production
• Commonly blooms in the mid-Atlantic states,
  permanently destroyed important shellfisheries in
  Long Island Sound.
• 2008 one of the worst blooms to date
• Can reach uncommonly high cell densities
• First whole genome sequence for a HAB species!

1http//ccmp.bigelow.org 2http//siddall.info/fift
een/bt.html 3http//www.scottsbt.com/misc/enviro/b
rowntide.html
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Two modes of harmful impacts from blooms

• 2 Non-Toxic effects
• Toxic effects
• Direct human impact through direct exposure
• Indirect toxin impacts organisms on its
  passage through the food web via trophic transfer

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Different HAB impacts

• Neurotoxic Shellfish Poisoning (NSP)
• Amnesic Shellfish Poisoning (ASP)
• Diarrhetic Shellfish Poisoning (DSP)
• Azaspiracid Shellfish Poisoning (AZP)
• Pfiesteria complex
• Ciguatera Fish Poisoning (CFP)
• Paralytic Shellfish Poisoning (PSP)
• Freshwater toxins

Karenia
Pseudoniztschia
Pfiesteria
Alexandrium
NSP
ASP
Pfiesteria complex
PSP
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Neurotoxic shellfish poisoning (NSP)

• Karenia brevis
• Athecate dinoflagellate
• Motile
• Brevetoxin (direct impacts)
• Commonly blooms in Florida, responsible for
  manatee deaths in 1996-1997 and recurrent fish
  kills that litter the beach.
• Bloom reports have doubled in the last 30 years

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Amnesic shellfish poisoning (ASP)

• Pseudonitzschia australis
• Diatom
• Non-motile
• Domoic acid (Indirect impacts)
• Common along west coast. California blooms
  responsible for sea lion deaths in 1999 and
  recurrent shell-fish contamination.

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Domoic acid can accumulate in the food web
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Paralytic shellfish poisoning (PSP)

• Alexandrium fundyense, A. tamarense and A.
  catenella
• Thecate dinoflagellate
• Motile
• Forms cysts
• Saxitoxin (indirect impacts)
• Common globally in temperate waters. Present and
  dangerous in the Pacific Northwest, and New
  England. Responsible for recent human poisonings
  the deaths of marine mammals.

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HAB seem to be getting worse

• Compared to 30 years ago, we have
• more algal toxins
• more toxic algal species
• more fisheries resources affected
• more areas affected
• higher economic costs
• A SERIOUS PROBLEM

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Possible mechanisms for the expansion of HAB

• Species dispersal or introduction via natural
  currents, storms cyst deposition
• More scientists, improved chemical analysis,
  better communication
• Increased aquaculture
• Dispersal by human activities (ballast water,
  shellfish seeding)
• Nutrient enrichment pollution

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Outline

• Introduction to Microbial Oceanography
• What are Harmful Algal Blooms?
• Who is out there?
• Detecting Alexandrium catenella in the Puget
  Sound
• What are they doing?
• Detecting alkaline phosphatase activity

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Who is there?

• How do we detect specific microbial
  populations????

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Similar morphology is a challenge!

• Toxic Alexandrium can cause PSP at low cell
  density.
• Toxic/Non-toxic Alexandrium appear very similar.

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Identification and enumeration qPCR
Primers
DNA
X
X

• Test for the Alexandrium catenella gene
  signature

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Standard curve

• Multiple strains of local Puget Sound Alexandrium
  catenella are used to construct a standard curve.

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PSP in Puget Sound

• PSP in the Pacific Northwest has been known for
  centuries (Trainer et al. 2003)
• Captain Vancouver 1793 (British Columbia)
• WA State Dept. Health (WDOH) initiated shellfish
  monitoring in the 1930s
• Several thousand PSP assays in mussels collected
  by volunteers from key sentinel sites each year
• No concurrent monitoring for Alexandrium
  catenella - the cause of PSP

Puget Sound WA
maps.google.com
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Key questions

• Was there toxicity in 2006?
• How few cells can we detect?
• Can we detect cells before shellfish toxicity?
• Does increasing cell number indicate lead to an
  increase in toxicity?
• What can we learn about Alexandrium catenella
  bloom dynamics?

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Sampling system
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Volunteers
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Samples arrive in Woods Hole
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Catalog the samples
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Extract DNA
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Prepare samples and standards for qPCR
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Run qPCR
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Run qPCR
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Tabulate data and determine cell numbers

• With volunteer help we analyzed nearly 600 DNA
  samples in 2006!

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Key questions

• Was there toxicity in 2006? - Yes
• How few cells can we detect?
• Can we detect cells before shellfish toxicity?
• Does increasing cell number indicate lead to an
  increase in toxicity?
• What can we learn about Alexandrium catenella
  bloom dynamics?

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Key questions

• Was there toxicity in 2006?
• How few cells can we detect? - Very few the assay
  is very sensitive!
• Can we detect cells before shellfish toxicity?
• Does increasing cell number indicate lead to an
  increase in toxicity?
• What can we learn about Alexandrium catenella
  bloom dynamics?

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Key questions

• Was there toxicity in 2006?
• How few cells can we detect?
• Can we detect cells before shellfish toxicity?
  Cells were detected at all stations before the
  legal saxitoxin limit.
• Does increasing cell number indicate lead to an
  increase in toxicity? - Yes - at some stations
• What can we learn about Alexandrium catenella
  bloom dynamics?

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Key questions

• Can we detect cells before shellfish toxicity?
• Does increasing cell number indicate lead to an
  increase in toxicity?
• What can we learn about Alexandrium catenella
  bloom dynamics? Seasonal patterns are interesting
  - but more work is required to identify what
  factors cause Alexandrium catentella to bloom in
  this system. (e.g. prediction is difficult)

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What are they doing?

• How do we the activities of specific microbial
  populations????

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What are they doing?

• What nutrients are important and when?
• Nutrients like phosphorus can come in inorganic
  and organic forms
• The cells typically preferentially use the
  inorganic form (PO4)
• The enzyme alkaline phosphatase is required to
  use organic phosphorus.

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Decreased toxicity on some organic nutrients
Organic
Inorganic
Dyhrman 2003
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Linking nutrients to bloom dynamics
Dyhrman 1999
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Key questions

• Do HAB use organic phosphorus? YES
• Prorocentrum minimum alkaline phosphatase
  activity
• Can blooms be controlled by phosphorus supply?
• Phosphorus stress in Narragansett Bay.

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Alkaline phosphatase activity
Dyhrman 1999
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Key questions

• Do HAB use organic phosphorus? YES
• Prorocentrum minimum alkaline phosphatase
  activity
• Can blooms be controlled by phosphorus supply?
  YES
• Phosphorus stress in Narragansett Bay.

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Inorganic phosphate concentration and the bloom
-P
P
Dyhrman 2001
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Summary

• Marine microbes are important!
• A very few microbes cause HAB - but they can lead
  to serious problems with toxins in the food web
• Molecular identification of DNA signatures is
  improving HAB detection and monitoring.
• New biochemical tools allow us to identify the
  activities of HAB and trace the connections
  between HAB events and nutrients.

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Outreach

• How do you engage kids (8-12) with scientific
  content on a broad scale? - Whyville, a virtual
  world for kids www.whyville.net

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