Chemical Cues in the Ocean Julia Kubanek Assistant Professor - - PowerPoint PPT Presentation

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Chemical Cues in the Ocean Julia Kubanek Assistant Professor - - PowerPoint PPT Presentation

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Chemical Cues in the Ocean Julia Kubanek Assistant Professor School of Biology and School of Chemistry & Biochemistry Organisms of all types are under natural selection pressure to avoid becoming food for others Some do this with

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Chemical Cues in the Ocean

Julia Kubanek Assistant Professor School of Biology and School of Chemistry & Biochemistry

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Organisms of all types are under natural selection pressure to avoid becoming food for others…

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Some do this with chemical defenses…

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Corals, marine sponges, worms, seaweeds face heavy predation pressure

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But herbivory and predation are actually healthy for coral reefs…

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Coral reefs are under threat worldwide… Probable factors include loss of herbivores, rising temperatures, storms, pollution, disease

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Historically, predation has been intense – and so prey have evolved various anti-predatory defenses

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Marine sponges: great candidates for testing hypotheses re anti-predator chemical defenses…

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Unpalatable compounds in sponges cause fish to reject food

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O COOH O O O O O O O OH HO OH OH OH OH OH OH OH OH

10 20 30 40 50 60 70 80 90 100 TREATED CONTROL PERCENT EATEN

CRUDE EXTRACT N = 20 (20), p < 0.0001

Unpalatable compounds in sponges cause fish to reject food

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20 40 60 80 100 TREATED CONTROL % EATEN n = 20, p = 0.0003

20 40 60 80 100 TREATED CONTROL n = 20, p = 0.011

Erylus formosus Ectyoplasia ferox

Predator deterrents in sponges

O COOH O O O O O O O OH HO OH OH OH OH OH OH OH OH

HO O COOH CH2OH O HO O O OH HO O OH OH OH O OH OH HO OH

Kubanek, Pawlik, Eve, Fenical (2000) Mar Ecol Prog Ser 207:69-77 Kubanek, Pawlik, Fenical (2001) Nat Prod Lett 15:275-285

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We know…

saccharideO COOH sacchO COOH Osacch sacchO COOH Osacch

2 4 6 8 10 0.1 1 10 100 Concentration (mg/mL) Pellets eaten of 10

  • that chemical defenses affect different predators

differently

  • concentration-dependence of individual compounds
  • that rejection occurs within ~1 sec or less

– involvement of specific chemoreceptors?

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gel treated with sponge triterpene glycosides

control gel

Some sponge compounds also deter settlement by algae and invertebrates

O COOH O O O O O O O OH HO OH OH OH OH OH OH OH OH

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Control gel Gel treated with sponge triterpene glycosides

Others sponge compounds deter overgrowth by neighboring sponges

Kubanek, Whalen, Engel, Kelly, Henkel, Fenical, Pawlik (2002) Oecologia 131:125-136

HO O COOH CH2OH O HO O O OH HO O OH OH OH O OH OH HO OH

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O COOH O O O O O O O OH HO OH OH OH OH OH OH OH OH

HO H H CN

O OMe O OMe O OH O OMe N O N O N O N OHC Me

Br HO Cl

Chemical defenses are common among marine plants and invertebrate animals

O NH O N N O O O OMe N H HN O O N O N O OH OH O O O

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Pathogens are poorly understood but clearly affect coral reef communities

unknown disease decimated this herbivore in 1980’s and led to widespread algal overgrowth of corals

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Marine plants can also suffer from disease

1930’s eelgrass wasting epidemic removed ~90% of North Atlantic eelgrass – recovery took decades

wasting disease on eelgrass coralline lethal orange disease

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Clod

Disease can cause losses

  • f commercially important algal species
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Seawater is full of potentially pathogenic microbes – why aren’t all seaweeds susceptible?

  • natural antibiotics protect some seaweeds?
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Testing seaweeds for antimicrobial chemical defenses

seaweed extraction 1) incorporation of seaweed extract into agar wells 2) inoculation with marine fungus 6 days for fungal growth comparison with no-extract controls

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ANTIMICROBIAL POTENTIAL OF 53 SEAWEEDS 53 seaweed species collected in the Bahamas and tested against marine fungi and bacteria collected in same environment

  • Over half of seaweed extracts deterred

growth by at least 1 microbe

  • Most showed specific activities
  • YES, antimicrobial chemical defenses seem to

be common and diverse

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Lobophora variegata

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BIOASSAY-GUIDED FRACTIONATION

liquid-liquid partitioning

hexanes chloroform ethyl acetate n-butanol water-soluble 10-20% aqueous methanol eluted fraction

reversed phase silica size exclusion chromatography

fractions 1-5 fractions 6-8 fractions 9-30 brown pigments orange and yellow pigments green pigments and galactolipids fractions 5-6

reversed phase HPLC

broad peak

repetitive normal phase HPLC

  • ne antifungal compound

lobophorolide [approx. 200 μg (0.0002 % by dry weight)]

Lobophora variegata extract

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LOBOPHOROLIDE STRUCTURE

O OMe O OMe OMe O O OH OMe OH O OMe

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LOBOPHOROLIDE IN THE BAHAMAS AND FLORIDA (73 samples)

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BIOMEDICAL POTENTIAL OF LOBOPHOROLIDE

Type of Bioactivity Assay Organism Bioactivity (μg/mL) Antifungal Candida albicans MIC = 1.3

  • C. albicans (amphotericin-resistant)

MIC = 0.5 Anticancer human colon tumor (HCT-116) IC50 = 0.03

Kubanek, Jensen, Keifer, Sullards, Collins, Fenical (2003) PNAS 100:6916-6921

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Drugs from the sea?

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Drugs from the sea

O O HO Br R OH Br O O HO OH Br Br Br O O HO OH Br Br Br O O HO Br OH Br 1 R=Br 2 R=OH 6 8 4 O O HO Br OH O 5

1 14 10 7 5 3 25 24 23 22 19 18 16 15 26 27 1 3 5 7 10 14 15 16 18 19 22 23 24 25 26 27

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1 3 5 7 10 15 16 18 19 21 22 23 24 26 25 27

O O HO OH O H O O HO Br Br OH Br 3 O O HO OH Br OH Br 9 O O HO OH Br OH Br 7 H H

Callophycus serratus from Fiji coral reefs Kubanek, Prusak, Snell, Giese, Hardcastle, Fairchild, Aalbersberg, Raventos-Suarez, Hay (2005) Org. Lett. 7:5261-5264 Kubanek, Prusak, Snell, Giese, Fairchild, Aalbersberg, Hay (in press) J. Nat. Prod.

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The Plankton…

  • Greatest source of fixed carbon and the source
  • f much of Earth’s biologically available nitrogen
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The Plankton…

  • Greatest source of fixed carbon and the source
  • f much of Earth’s biologically available nitrogen
  • Abundance of grazers (copepods, rotifers, etc.)

plus microbial diversity: complex population and community interactions

  • “Featureless” – large scale physical patterns of

nutrients, temperature, light, fluid flow

  • vs. small phytoplankton & zooplankton
  • How important are chemical cues?
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RED TIDES: BLOOMS OF (TOXIC?) PHYTOPLANKTON

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Direct costs to U.S. fisheries, tourism, monitoring, human health: $20M per bloom

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Human health effects of red tides

  • Phytoplankton toxins cause

gastrointestinal and neurological symptoms

  • 3 modes of exposure

– consumption of filter feeding bivalves – concentrated through food web to fish – inhalation of airborne toxins

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domoic acid brevetoxin B (PbTx-2) ciguatoxin (CTx-1) saxitoxin

POTENT NEUROTOXINS PRODUCED BY PHYTOPLANKTON

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  • Phytoplankton neurotoxins don’t have the same

effects on all organisms

  • Not clear how neurotoxins affect predators

and competitors

  • How can some phytoplankton bloom

at a million cells per liter or more? – do they use toxins to escape predation?

  • to exclude competitors?
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Hypothesis:

Red tide phytoplankton are engaging in chemical warfare against other phytoplankton

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MODEL SYSTEM: FLORIDA RED TIDE DINOFLAGELLATE Karenia brevis

brevetoxin B (PbTx-2)

O O O O O O O O O O O O Me H Me H H Me Me H H H Me H H H H H Me H Me H H H HO CH2C(=CH2)CHO

Kubanek, Hicks, Naar, Villareal (2005) Limnol. Oceanog. 50:883-895

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Growth of competing phytoplankton is suppressed by red tide cells and by red tide exudates

alone + red tide exudate + red tide cells n=5

Asterionellopsis glacialis

3 5 8 10 1.0×105 2.0×105 3.0×105

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Time (days) Cells per mL

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Strong evidence for red tide chemical warfare against several competitors

Skeletonema costatum

5 10 15 20 25 45 95 145

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TIme (days) Fluorescence (μg/L chla)

Prorocentrum minimum

10 20 30 40 50 50 100 150

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Time (days) Fluorescence (μgchla/L)

Asterionellopsis glacialis

5 10 15 20 25 30

  • 5

5 15 25 35 45 55

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Time (days) Fluorescence (μg/L chla)

Akashiwo sanguinea

5 10 15 20 5 10 15

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Time (days) Fluorescence (μg/L chla)

extracts of red tide in stationary phase extracts of red tide in logarithmic phase control

Emily Prince, unpublished

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Red tide brevetoxins suppress competitor Skeletonema costatum

Skeletonema costatum

0.0 2.5 5.0 7.5 10.0 12.5 15.0 50 100 150 200

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Time (days) Fluorescence (μg/L chla)

control brevetoxins

O O O O O O O O O O O O Me H Me H H Me Me H H H Me H H H H H Me H Me H H H HO CH2C(=CH2)CHO

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Brevetoxin concentration per red tide cell

1 2 3 4 5 6 7 8 9 10 10 20 30 40 50

red tide alone red tide + Skele Time (days) Concentration (pg/cell)

Skele fights back against red tide by inhibiting brevetoxin production

Tracey Myers & Emily Prince, unpublished

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O O O O O O O O O O O O Me H Me H H Me Me H H H Me H H H H H Me H Me H H H HO CH2C(=CH2)CHO

10 20 30 40 50 60

a b b

Treatment brevetoxin concentration (pg per red tide cell)

red tide alone red tide cultured with Skele cells red tide cultured with extracts of Skele

Skele produces a chemical inhibitor

  • f red tide brevetoxins

We are working to identify this inhibitor…

Emily Prince & Tracey Myers, unpublished

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Summarizing plankton chemical warfare:

  • Florida red tide poisons its neighbors
  • several (unknown) red tide compounds act against several

competitors

  • red tide brevetoxins suppress one important competitor

(Skele)

  • Skele fights back by inhibiting brevetoxin production by

red tide cells

  • interactions among planktonic organisms are complex!
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Overall conclusions

  • chemical cues are crucial in competitive, predator-prey,

and host-pathogen interactions

  • the molecules involved are structurally complex, regulated

by subtle communication cues, and have specific targets

  • the long-term persistence of populations and species, and

therefore ecosystem function, is driven by these interactions

  • we may be able to co-opt marine organisms’ defenses and
  • ffenses in the search for new and better drugs
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Acknowledgments

Present and past group members:

Emily Prince Tracey Myers Melissa Hicks Anne Prusak Amy Lane Ruth Armour Paige Auten Rachel Giese Katie McCurdy Doug Young Kristen Whalen

  • Dr. Dwight Collins
  • Dr. Michiya Kamio

Collaborators:

Tracy Villareal (UT) Jerome Naar (UNCW) Bill Fenical (Scripps) Joe Pawlik (UNCW) Mark Hay (GIT) Terry Snell (GIT) Bill Aalbersberg (Fiji) Ken Hardcastle (Emory) Craig Fairchild (Bristol-Myers Squibb)

Funding:

National Science Foundation National Institutes of Health Camille & Henry Dreyfus Foundation Georgia Tech Blanchard Assistant Professorship