Distribution of the Northern Star Coral Distribution of the Northern Star Coral ((Astrangia poculataAstrangia poculata) and its Symbiotic ) and its Symbiotic Zooxanthellae on New Jersey Artificial Zooxanthellae on New Jersey Artificial

Reefs.Reefs.

Peter F. Straub, Professor of Biology, DSOTara Harmer, Assistant Professor of Biology

School of Natural Sciences & MathRichard Stockton College of NJ

Collaboration-Undergraduate Collaboration-Undergraduate Research and TeachingResearch and Teaching

Stockton Scientific Diving Stockton Scientific Diving Program- Straub -DSOProgram- Straub -DSO

BIOL 2175- Scientific BIOL 2175- Scientific Diving-Straub Fall 07Diving-Straub Fall 07

BIO 4211- Molecular BIO 4211- Molecular Evolution- Harmer Fall 07Evolution- Harmer Fall 07

BIOL 4215-Biotechnology- BIOL 4215-Biotechnology- Straub, Spring 08Straub, Spring 08

BIOL 4800- Biology BIOL 4800- Biology Research- Harmer Research- Harmer Fall/Spring 07/08Fall/Spring 07/08

Links

BIOL 2175 Fall 2007

Astrangia poculata Astrangia poculata (syn. (syn. A.A. danaedanae)) -Northern Star Coral -Northern Star Coral

Cnidaria › Anthozoa › Hexacorallia › Scleractinia › Faviina › Cnidaria › Anthozoa › Hexacorallia › Scleractinia › Faviina › Rhizangiidae › Astrangia Rhizangiidae › Astrangia

Found from the sub-tropics to the temperate zone <5C. Most corals Found from the sub-tropics to the temperate zone <5C. Most corals live in tropical to sub-tropical waters above 20 C and cannot tolerate live in tropical to sub-tropical waters above 20 C and cannot tolerate high turbidityhigh turbidity

Colonies can be encrusting, massive (mounding) or branching Colonies can be encrusting, massive (mounding) or branching

The individual coral skeletons or corallites are well defined, circular, The individual coral skeletons or corallites are well defined, circular, compact, and up to 10 mm in diametercompact, and up to 10 mm in diameter

Living coral tissue may be translucent to dark brown depending on the Living coral tissue may be translucent to dark brown depending on the presence and number of zooxanthellae presence and number of zooxanthellae

May contain zooxanthellate, or not, given local light conditions. May contain zooxanthellate, or not, given local light conditions.

http://www2.aims.gov.au/coralsearch/html/

HypothesisHypothesis

Since the distribution of northern star Since the distribution of northern star coral in NJ is patchy, only found on coral in NJ is patchy, only found on hard surfaces such as wrecks, is hard surfaces such as wrecks, is there any detectable genetic there any detectable genetic differentiation among local collection differentiation among local collection sites and between disjunct sites and between disjunct populations.populations.

MethodsMethods Site selectionSite selection Collect corals by scubaCollect corals by scuba Extract chromosomal DNAExtract chromosomal DNA Amplify ssRNA (18S) sequence by PCRAmplify ssRNA (18S) sequence by PCR DNA SequenceDNA Sequence Align sequences (Vector nTi) Align sequences (Vector nTi) Look for relatedness in coral pops Look for relatedness in coral pops

(MacClade and PHYLIP)(MacClade and PHYLIP)

Coral Collection sites7 sites, 15 dives

NJ

Lemuel Burrows, torpedoed, 3/14/1942, 80 ft water

Jet Trader, yard oiler, 95 ft., artificial reef, sunk 9/2/05

Car float, 60 ft

John MarvinClam DredgeSunk 1/16/199270 ft.

http://njscuba.net/sites

Wreck of the Almirante (the flour wreck) off Wreck of the Almirante (the flour wreck) off Atlantic City NJ (approx 8 mi) at 70 ft. Atlantic City NJ (approx 8 mi) at 70 ft.

United Fruit Co. vessel (378 ft x 50 ft) sunk in United Fruit Co. vessel (378 ft x 50 ft) sunk in collision Sept 6, 1918 w/ Navy tanker USS Hisko, collision Sept 6, 1918 w/ Navy tanker USS Hisko,

later wire dragged 2x, depth charges, fairly later wire dragged 2x, depth charges, fairly broken upbroken up

Underwater photos David Roche

http://njscuba.net

Coral collections 8/25/07, 8/29/07 and 10/6/07

Northern Star CoralAstrangia poculata

Sea Girt, Clam Dredge, Depth 70 ft sunk 8/1990. Coral Collection- 7/28/07

Photos Pete Straub

MV Atlantus Charters

Almirante (Flour)

Mike

Dave

Pete

Almirante (Flour)

Almirante

Coral plus mussels

Coral plus anemoneCoral heads Coral heads

Photos P. Straub & T. Harmer

Coral polyps extended

Individual coral polyps

Photos P. Straub & T. Harmer

Kept 10 gallon aquaria, filtered,Feed zooplankton, commercialzooplankton (preserved).

DNA Extractions

Remove tissue from single corallite using scalpel blade tip (like cutting grapefruit)

Qiagen DNAeasy (column method) Kits

Eluted DNA from columns with Sterile water

Measure DNA content at A260 with spectrophotometer

Analyze by agarose gel electrophoresis- High MW

Adjust concentration for PCR reaction

1. PCR Reaction Conditions100 ng chromosomal DNAQiagen TAQ polymeraseQuiagen coraload buffer0.2 uM dNTP’sForward Primer NS1*Reverse Primer Euk 516R**

1 cycle 94 C, 1:00 min35 cycles 94 C, 1:00 min 52 C, 1:30 min 72 C, 2:00 min 1 cycle 72 C, 10:00 min

Polymerase chain reaction (PCR)w/universal eukaryotic small subunit (ss)RNA- (18S)

~550 bp

*White et al., 1991 **Di’ez et al., 2001

3. Purify PCR products using Promega SV gel and PCR product cleanup colums kit

2. Analyze by agarose gel electrophoresis

Cloning amplified products from Coral PCR• Purified coral PCR products were cloned using a pGemT cloning kit (Promega kit) simplifies cloning as PCR products may have an “A” base overhang and is simple to ligate to pGemT plasmid vector. • Ligation products were transformed into E. coli.• E. coli colonies were selected on ampicillin.• DNA plasmid minipreps were performed on individual E. coli cultures•Plasmids concentration measured by spectrophotometer A260

Automated Fluorescent DNA Sequencing• Use ~200 ng coral plasmid DNA, Beckman Coulter DCTS master mix With forward (T7) and reverse (sp6) sequencing primers

• Separation Beckman Coulter CEQ 8000 Genetic Analyzer

• Export sequence trim out plasmid vector sequences