Hematodinium in the Context of Other Dinoflagellate Populations in the MCBs
Hematodinium sp.
First described as a parasite of the green crab (Carcinus maenas) during the 1930’s off the French Coast
Invades hemolymph of host; consumes hemocyanin and
mobilized host metabolites; hemocyte numbers reduced
Combined lack of oxygen, nutrition, tissue damage leads to crab morbidity and/or death
Nagle, Place, Schott, Jagus, Messick, and Pitula; Diseases of Aquatic Organisms. 84:79-87, 2009.
Large Host Range in Diverse Ecosystems
Blue crab (Callinectes sapidus)
-Eastern and Southern US coastal waters
Edible Crab (Cancer pagurus)
-Northern Atlantic and Bay of Biscay
Snow Crab (Chionoectes opilio)
-Alaskan waters, Newfoundland Bay
Tanner crab(Chionoecetes bairdi)
-Alaskan waters
Blue swimmer crab (Portunus pelagicus)
-Moreton Bay, Australia
The Norway lobster (Nephrops norvegicus)
-Northeastern Atlantic
Ridgetail white prawn shrimp (Exopalaemon carinicauda)
-Aquaculture ponds in East China Sea
Various amphipods
- Eastern and Southern US coastal waters
Proposed Life Cycle in the Blue Crab
How do blue crabs become infected?
a) Physical- Blue crab eats infected crabs or intermediate host
b) Dinospore- stage in water possibly encapsulated in a
sporocyst. Acquired through gills?
http://www.americanscientist.org/templat
e/AssetDetail/assetid/37182/
page/2;jsessionid=aaa5LVF0#37425
Dinospores Are Emitted from Diseased Crustaceans
Motile cells observed following death
of a crab with Hematodinium.
Dr. Sue Marrs in Stentiford and Shields DAO,
2005, 66: 47-70 http://www.vims.edu/~jeff/biology/stentiford%20and%20shields%202005.pdf
Cancer pagurus
Kim Jennings at IMET (Schott and Jagus lab)
2006 LMRCSC Internship
Messick and Shields 1994, Diseases of Aquatic Organisms
Hematodinium sp. Infection in Blue Crabs Has Seasonal Cycles
Goals of Our Lab
Very little is known about Hematodinium disease
transmission in nature
Find potential hotspots of Hematodinium in the Maryland
Coastal Bays (MCB)
a) Analyze its presence according to seasonal cycles
b) Uncover ecological variables that are associated with free-
living Hematodinium, and also discover potential alternate hosts
Chesapeake Bay
UMES CETRC/
NPS Chincoteague
Bay
Searching for Hematodinium Reservoirs
Collaboration with National Park Service in search of
Hematodinium sp. reservoirs in water and sediment samples.
Historically the coastal bays have shown high infection rates.
•Monthly monitoring sites of
the National Park Service
(blue ellipses).
•Surveys of the NPS sites
were conducted from
April-November of 2010
and 2011.
•All sites have an excellent
historical record through
NPS and MDDNR for
temperature, dissolved
oxygen, pH, and turbidity.
Commercial Harbor
O.C Inlet
Snug Harbor
Verrazano Bridge
South Point
Cedar Island
Whittington Point
Wildcat point
Assateague Channel
Tom’s Cove
Trappe Creek
Newport Bay
Johnson’s Bay
Taylor’s Landing
Greenbackville
Sinnickson
Chincoteague Channel
ITS1
TCGCACGAAGAAAATAATAATATATTTTATTATTTTCGCACACAAACATTCACCGTGAACCTTAGCCATTAGCTAC
GACGACTACTAGCTAGCTACTGAGTGGGGCGGTGGTGTGTTGGTTACTACTGCTACTTCTTACTCGTAGCTGA
ACTGCACACACACTAGTACCCCTCTCTTGCTGGTAGGAGAAGTAGCTTCTACGGGGTGTGAGGGTACGGTGG
TAGTACACGCCTACCACTGAACTCCTCCATCCCACGTTTGCTTTCCATAAACACAACATCTCTAATTTCAGCTAT
TCATCTTGCTCTGCTCCCTTTCGCGGGGATAGGGCTTTCTTCAAACGTATGAC
5.8S
TAGAAAATTTTAGCGATGAATGCCTCGGCTCGGGTTACGATGAAGGACGCAGCGAATTGCGATAAGCAATGCG
AATTGCAGAATTCCGTGAATCATCAGATTTTTGAACGTACTCTACGCTCTCGGGTATCCCTGGGAGCATGTCTG
GTCTCAGC
ITS2
GTCTGTTCAACCTTTTGTGCCTCCTGGAGTTGTGAACATTCTCCTTCTTGGAAGCGATTTTGTGCACCAGTGA
GCCTCTTTCCACACACATGCTCTACGACGCCTTGTTGTTGTAGACAGCGGAAGATGGCCATTGACGCATTAAA
TATTAAGGGATTTGTAGAATGTTGTAGAGAGGGTTGGTTGCGTACGTCTCACCGTACGCACCAAAAGCTCTGC
ATGTTCCCCAACAACACTTATGACCCACTTTAGGTCTAATGCTTGTTGGCCGAAGGGTTACACTGCATGGTTAT
ACCGCTACTCTTCTTCCGCCCTTTACCGTGATAGTACACAGGTTTTCGGACTAGTGGCGCTATTGCAGCAGAA
ATATTTATATCTCTGTATATATTTACACATG
SSU
ITS2
5.8S
ITS1
NTS NTS LSU
Water Collection Dates Sediment Collection Dates
Site 1 Commercial Harbor 7_10 8_11
Site 2 Verrazano Bridge 9_10 4_10; 5_10; 7_10; 8_11
Site 3 Newport Bay 6_10; 9_10 6_10; 8_11
Site 4 Trappe Creek 4/10; 7/10 8_11
Site 5 Public Landing 6_10; 9_10 5_10; 8_11
Site 6 Whittington Point 8_10; 8_11
Site 7 Taylor's Landing 4/10; 6/10; 7/10
Site 8 Wildcat Point 8_10; 8_11
Site 9 Greenbackville 6_10 8_11
Site 10 Sinnickson
4_10; 6_10; 7_10; 8_10;
10_10 5_10; 7_10; 8_10; 10_10; 11_10
Site 11 Chincoteague Channel 7_10
Site 13 Tom's Cove 6_11 8_10; 10_10; 8_11
Site 14 Johnson's Bay 4_10; 6_10
Site 15 Cedar Island 7_10
Site 18 Snug Harbor 10_10 4_10; 6_10
Endpoint PCR Analysis for Environmental Detection
48 of 546 (8.8%) of environmental samples from the Maryland and Virginia
coastal bays were positive for Hematodinium sp.
Four sites had detectable signal in water in April: earliest detection date we
are aware of.
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*
*
*
Hematodinium sp. “Hotpsots” 2010 2011
Water Sediment Water Sediment
April Trappe Creek Verrazano Bridge Taylors Landing Snug Harbor Sinnickson Johnson’s Bay May Verrazano Bridge Public Landing Sinnickson June Newport Bay Newport Bay Tom’s Cove Public Landing Snug Harbor Taylors Landing Greenbackville Johnson’s Bay July Commercial Harbor Verrazano Bridge Trappe Creek Sinnickson Taylor’s Landing Chincoteague Channel
Cedar Island August Whittington Point Commercial Harbor Wildcat Point Verrazano Bridge Sinnickson Newport Bay Tom’s Cove Trappe Creek Public Landing Tom’s Cove Whittington Point Wildcat Point Greenbackville Sept. Verrazano Bridge Newport Bay Public Landing Oct. Sinnickson Sinnickson Snug Harbor Nov. Tom’s Cove
Figure Courtesy Darlene Wells (MD-DNR)
and Roman Jessien (MCBP)
“hotspots” all correlate with higher
clay % soils
Water Gene Copies/ml
Sinnickson Apr 2010 2.1 X107
Newport Bay September 2010 6.2 x 105
Sinnickson October 2010 2.6 x 105
Tom's Cove June 2011 8.1 X 107
Sediment Gene Copies/g Sediment
Trappe Creek April 2010 1.7 x 105
Sinnickson May 2010 3.0 x 105
Newport Bay June 2010 4.5 X 105
Sinnickson July 2010 1.7 x 105
Trappe Creek July 2010 1.4 X 105
Sinnickson August 2010 2.6 x 107
Tom's Cove August 2010 1.1 x 107
Newport Bay August 2010 2.4 x 105
Trappe Creek August 2010 3.2 x 105
Newport Bay September 2010 3.9 x 107
Sinnickson October 2010 2.3 x 106
Tom's Cove November 2010 1.2 x 106
Quantification of High Incidence Locations by QPCR
In water samples, highest parasite levels were observed in spring months
(not what we hypothesized)
In sediment samples, highest parasite levels were in the late summer
(consistent with what we hypothesized)
Hematodinium Other species
April 15/16 1 unidentified nanoflagellate June 2/13 10 Heterocapsa rotundata, 1 Peridinium sp.
July/August 13/25 3 Gymnodinium sanguineum, 1 Gymnodinium sp., 3 H. rotundata
and 5 unidentified dinoflagellates
October 10/16 2 Pentapharsodinium tyrrhenicum, 1 G. simplex, 1 Gymnodinium sp.,
1 H. rotundata and 1 Dinophyceae sp. 1
Analysis of Hematodinium sp. at Sinnickson, Through
Clone Libraries Analysis of Dinoflagellate 18 S rRNA
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Chl Chl
(ug/L) (ug/L)
Newport Bay-3 April 8.2 3.9
May 8.8 17.1
June 20.02 9.4
July 22.7 15.3
Aug. 25.9 16.8
Sept. 9.4
Oct. 14.7 8.3
Nov. 8.7 6.8
Trappe Creek-4
April 9.5 4.2
May 13 18
June 18.7 23.8
July 39.6 27.3
Aug. 28 27.1
Sept. NA 8.1
Oct. 12.3 9.5
Nov. 10.5 8.7
Public Landing-5
April 7.2 2.2
May 19.4 9.2
June 23.1 9.7
July 24.4 4.5
Aug. 15.4 9.3
Sept. 6
Oct. 19.2 3.6
Nov. 4 5.8
Taylor's Landing-7
April 5.4 4.9
May 9.8 8.9
June 23.4 8.1
July 21.7 11.5
Aug. 28.3 14.6
Sept. 6.2
Oct. 12.8 4.5
Nov. 3.3 2.7
In Summary…
a) We have detected in the water column and sediment samples from the
MD and VA Coastal Bays. About 9% of samples were detected positive.
b) Hematodinium sp. DNA is present in the environment throughout all
seasons although there was a surprising amount detected in early
spring, particularly in water
c) We have the expertise to monitor both for Hematodinium disease and
also for the relationships among resident dinoflagellates, including
potential HAB-forming species
Future Directions a) Quantify Hematodinium in environmental reservoirs using QPCR
b) Understand the nature of the “free-living” samples. Ie. Are they infective
dinospores? Are they reproductive forms? Are they in association with
zooplankton?
c) Is there a seasonal succession pattern in the development of HABs?
National Park
Service
Brian Sturgis
Eric Sherry
UMCES-IMET
Feng Chen
Ihuoma Njoku
Eric Schott
Ammar Hanif
Acknowledgements