Does Stress Causes Changes In Bacterial Community Associated

With Soft Coral Plexaurahomomalla?

Zeenat TinwalaUniversity of Houston-Clear Lake

Houston- Texas

zeenat16@gmail.com

CORAL REEFS AND SOFT CORAL

• Most diverse ecosystems - Economically valuable – Food, fishing, tourism and costal protection

• Corals: marine invertebrates (animals), Phylum Cnidaria, Class Anthozoa

• Soft corals: lack calcium carbonate skeleton

• Producers of marine natural products (MNPs) with biological activity

Eunicea fusca:

fuscosides

Plexaura homomalla:

Prostaglandins

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CORAL STRESS: BLEACHING

• Corals - Stress

• Bleaching – Loss of symbiotic dinoflagellate Symbiodinium sp.

• Possible disruption of beneficial bacteria & pathogen susceptibility

• Prolonged bleaching - coral death Photo: Great Barrier Reef Marine Parks Authority

Bleaching Disease3

PROJECT AIM

• Aim: To identify bacterial species associated in the soft coral Plexaura homomalla during normal and stress conditions

• Culture-independent technique (CIT) - 16S rDNA clone library analysis

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• Non-stressed samples: Population of mostly

Proteobacteria, especially beneficial gamma-

Proteobacteria.

• Atypical bacteria such as other Proteobacteria

(especially pathogenic Vibrios), Firmicutes and

Actinobacteria expected in stressed organisms.

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HYPOTHESIS

Samples collected Summerland Key Reef, FL, June 2010

Three stress treatments (12 hours):

• Heat: 31 C

• LPS (Lipopolysaccharides): 10µg/ml

• Control (27 C)

Flash Frozen and stored at -80oC

DNA Isolation NucleoSpin Soil – Macherey-Nagel

PCR: 16S rDNA bacterial primers

Clone Library (TOPO TA vector)

RFLP (Restriction Fragment Length Polymorphism) HhaI and TaqI

DNA Sequencing & Analysis6

RFLP RESULTS

CONTROL

A A AA BB

500

100

bp

HEAT

A A A A AB B B B

100

bp

500

C D

LPS

A A A A AB

100

500

bp

B AA

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RESULTS

RFLP Pattern

# Control Clones

# Heat Clones

# LPS clones

Pattern A 7 6 10

Pattern B 4 6 1

Pattern C - 1 3

Pattern D 2 - 1

Pattern E - 1 -

Undefinable 6 5 4

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FUTURE WORK

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• Screen more clones

• Sequence samples with different RFLPpatterns

• Construct a phylogenetic tree with sequencedata

• Compare results from other culturedependent (classic plate culture), independenttechniques (DGGE) and to production ofprostaglandins

CONCLUSION

• Culture Independent techniques like RFLP and DGGE analysis are helpful preliminary basis in determining changes of bacteria in soft corals

• Studying the changes in microbial flora of the corals might allow the design of biomarkers for prediction of early stress

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ACKNOWLEDGEMENTS

• Dr. Lory Santiago-Vazquez for guiding and providing with the materials and lab space

• Research students Semhar Kesete & Catherine Sampson

• University of Houston-Clear Lake

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