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 chemical defenses…
Corals, marine sponges, worms, seaweeds face heavy predation pressure
But herbivory and predation are actually healthy for coral reefs…
Coral reefs are under threat worldwide… Probable factors include loss of herbivores, rising temperatures, storms, pollution, disease
Historically, predation has been intense – and so prey have evolved various anti-predatory defenses
Marine sponges: great candidates for testing hypotheses re anti-predator chemical defenses…
Unpalatable compounds in sponges cause fish to reject food
Unpalatable compounds in sponges cause fish to reject food 100 CRUDE EXTRACT 90 OH OH OH OH COOH N = 20 (20), p < 0.0001 O O O 80 O O O PERCENT EATEN OH OH O OH OH 70 O HO OH 60 50 40 30 20 10 0 TREATED CONTROL
Predator deterrents in sponges Ectyoplasia ferox Erylus formosus OH OH OH OH OH OH COOH OH COOH OH O O O O OH HO O O O O HO CH 2 OH O O HO OH OH O O OH OH OH O O HO HO OH OH 100 100 n = 20, p = 0.0003 n = 20, p = 0.011 80 80 % EATEN 60 60 40 40 20 20 0 0 TREATED CONTROL TREATED CONTROL Kubanek, Pawlik, Eve, Fenical (2000) Mar Ecol Prog Ser 207:69-77 Kubanek, Pawlik, Fenical (2001) Nat Prod Lett 15:275-285
We know… • that chemical defenses affect different predators differently • concentration-dependence of individual compounds • that rejection occurs within ~1 sec or less – involvement of specific chemoreceptors? 10 Pellets eaten of 10 8 6 4 2 0 0.1 1 10 100 Concentration (mg/mL) Osacch Osacch COOH COOH COOH sacchO sacchO saccharideO
Some sponge compounds also deter settlement by algae and invertebrates gel treated with sponge triterpene control gel glycosides OH OH OH OH COOH O O O O O O OH OH O OH OH O HO OH
Others sponge compounds deter overgrowth by neighboring sponges OH OH OH COOH OH O OH HO O HO CH 2 OH O O HO O OH O HO OH Gel treated with sponge Control triterpene gel glycosides Kubanek, Whalen, Engel, Kelly, Henkel, Fenical, Pawlik (2002) Oecologia 131:125-136
Chemical defenses are common among marine plants and invertebrate animals OH OH OH OH COOH O O O O O O OH OH O OH OH O HO OH OH OHC O O OH N O Me OMe O OMe O O O N O N O HN O O OH NH O O N O O O N O N N H N N O OMe O OMe CN H HO Br H HO Cl
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
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
Disease can cause losses of commercially important algal species Clod
Seawater is full of potentially pathogenic microbes – why aren’t all seaweeds susceptible? - natural antibiotics protect some seaweeds?
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
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
Lobophora variegata
BIOASSAY-GUIDED FRACTIONATION Lobophora variegata extract liquid-liquid partitioning hexanes chloroform ethyl acetate n -butanol water-soluble reversed phase silica 10-20% aqueous methanol eluted fraction 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 one antifungal compound lobophorolide [approx. 200 μ g (0.0002 % by dry weight)]
LOBOPHOROLIDE STRUCTURE O OMe O OMe OMe OMe OH O OH O OMe O
LOBOPHOROLIDE IN THE BAHAMAS AND FLORIDA (73 samples)
BIOMEDICAL POTENTIAL OF LOBOPHOROLIDE Type of Bioactivity Assay Organism Bioactivity ( μ g/mL) Antifungal Candida albicans MIC = 1.3 C. albicans (amphotericin-resistant) MIC = 0.5 Anticancer human colon tumor (HCT-116) IC 50 = 0.03 Kubanek, Jensen, Keifer, Sullards, Collins, Fenical (2003) PNAS 100:6916-6921
Drugs from the sea?
Drugs from the sea R Br 26 27 O O O 15 16 O 14 O O 1 25 3 18 HO OH HO OH HO OH 24 5 10 Br Br Br 7 23 Br Br Br 19 22 1 R=Br 3 4 2 R=OH OH Callophycus serratus O O 26 27 O from Fiji coral reefs 16 Br OH 1 15 O O O 14 3 HO 18 HO HO O 24 25 5 10 H OH 7 OH 23 Br Br Br Br Br 19 22 5 6 7 OH O O 26 27 O Br OH 25 O O 16 O 1 15 24 HO HO 18 3 HO 23 O 5 OH OH 10 Br Br Br Br H 7 22 H 19 21 8 10 9 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.
The Plankton… • Greatest source of fixed carbon and the source of much of Earth’s biologically available nitrogen
The Plankton… • Greatest source of fixed carbon and the source of 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?
RED TIDES: BLOOMS OF (TOXIC?) PHYTOPLANKTON
Direct costs to U.S. fisheries, tourism, monitoring, human health: $20M per bloom
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
POTENT NEUROTOXINS PRODUCED BY PHYTOPLANKTON ciguatoxin (CTx-1) domoic acid saxitoxin brevetoxin B (PbTx-2)
• 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?
Hypothesis: Red tide phytoplankton are engaging in chemical warfare against other phytoplankton
MODEL SYSTEM: FLORIDA RED TIDE DINOFLAGELLATE Karenia brevis HO Me CH 2 C(=CH 2 )CHO H O Me Me H Me Me H H O H O H O O O H O H O H O O H O O H H H H H Me Me brevetoxin B (PbTx-2) Kubanek, Hicks, Naar, Villareal (2005) Limnol. Oceanog. 50:883-895
Growth of competing phytoplankton is suppressed by red tide cells and by red tide exudates Asterionellopsis glacialis 3.0×10 5 alone Cells per mL 2.0×10 5 + red tide exudate * 1.0×10 5 + red tide cells * 0 0 3 5 8 10 Time (days) n=5
Strong evidence for red tide chemical warfare against several competitors Asterionellopsis glacialis Akashiwo sanguinea 55 15 45 Fluorescence Fluorescence ( μ g/L chla) 35 ( μ g/L chla) 10 * control 25 extracts of red tide in stationary phase 5 15 extracts of red tide in logarithmic 5 * * phase 0 -5 0 5 10 15 20 0 5 10 15 20 25 30 Time (days) Time (days) Prorocentrum minimum Skeletonema costatum 145 150 * Fluorescence Fluorescence * ( μ g/L chla) ( μ gchla/L) * 95 * 100 45 50 0 0 10 20 30 40 50 0 5 10 15 20 25 Emily Prince, unpublished Time (days) TIme (days)
Red tide brevetoxins suppress competitor Skeletonema costatum Skeletonema costatum 200 150 Fluorescence ( μ g/L chla) control * brevetoxins HO Me 100 CH 2 C(=CH 2 )CHO H O Me Me H Me Me H H H O O H O O O H O H O H 50 O O H O O H H H H H Me Me 0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Time (days)
Skele fights back against red tide by inhibiting brevetoxin production Brevetoxin concentration per red tide cell 50 red tide alone Concentration (pg/cell) red tide + Skele 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 Time (days) Tracey Myers & Emily Prince, unpublished
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