Anophryoides haemophila AH6

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Environmental Data
Principle Investigator(s)	Spencer Greenwood
External sample ID	Rick Cawthorn, Fraser Clark, Adam Acorn
NCGR Sample ID	MMETSP1019
Sample accession number	CAM_SMPL_002585
Assembly accession number	CAM_ASM_000403
Combined Assembly Name	N/A
Genus	Anophryoides
Species	haemophila
Strain	AH6
Clonal	Yes
Axenic	Yes
Prelim. NCBI Taxon ID	46462
18S rRNA	AACCTGGTTGATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTATAAAT AGTATACAGTGAAACTGCGAATGGCTCATTAAAACAGTTATAGTTTATTTGATAATGGAAAGCTACATGG ATAACCGTGGTAATTCTAGAGCTAATACATGCTGTCAAACCCGACTTTTTGGAAGGGTTGTATTTATTAG ATATTAAGCCAATATTCCTTGTGTCTATTGTGGTGAATCATAGTAACTGATCGAATCCCTTTTTGTGGAT AAATCATTCAAGTTTCTGCCCTATCAGCTTTCGATGGTAGTGTATTGGACTACCATGGCAGTCACGGGTA ACGGAGAATTAGGGTTCGGTTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCAG GCGCGTAAATTACCCAATCCTGATTCAGGGAGGTAGTGACAAGAAATAACAACCTGGGGGAATTTATTCC TTACGGGATTGCAATGAGAACAATTTAAACGACTTATCGAGGAACAATTGGAGGGCAAGTCTGGTGCCAG CAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAATT TCTGCGTATGCCTAGTTCTGGCTTTGGTCAAGCTAGTGTGTGCGCATCCGCTTGCAAATCCGGACCGGCG TTTACTCGTCGACCGGGGGAGTAGGCCCTTTACCTTGAAAAAATTAGAGTGTTTCAGGCAGGCAATGGCT CGAATACATTAGCATGGAATAATAGAATAGGACTTTTGTCTATTTGGTTGGTTATTAGACATGAGTAATG ATTAAAAGGGACAGTTGGGGGCATTAGTATTTAATAGTCAGAGGTAAAATTCTTGGATTTATTAAAGACT AACTTATGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTAGGGGATCAAAGACGA TCAGATACCGTCCTAGTCTTAACTATAAACTATACCGACTCGGAATCGGGCAGGCTTATAAATCTTGCTC GGCGCCGTATGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGA ATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTTACCAG GTCCAAACATGGGTGGGATTGACAGATTGAGAGCTCTTTCTTGATCCTATGGGTGGTGGTGCATGGCCGT TCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGAACGAGACCTTAACCTGCTAAATAGTAC GTTGATGCATAATTGGCGTTACTTCTTAGAGGGACTATGCGCTTTGAAACGCATGGAAGTTTGAGGCAAT AACAGGTCTGTGATGCCCTTAGATGTCCTGGGCCGCACGCGCGCTACAATGACTCACTCAGAAAGTATTT CCTGGCCCGGAAGGGTTCAGGGTAATCTTCATAATATGAGTCGTGTTAGGGATCGATCTTTGCAATTATA GATCTTGAACGAGGAATGCCTAGTAAGTGCAAGTCATCAGCTTGTACTGATTACGTCCCTGCCCTTTGTA CACACCGCCCGTCGCTCCTACCGATTTCGAGTGATCCGGTGAACCTTCTGGACTGAAGTCATTTATGACA TTGGGAAGTTAAGTAAACCTAATCACTTAGAGGAAGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAACCT GCAGAAGGATC
Importance of organism and transcriptomes	This is the only known ciliate pathogen of the American lobster (Homarus americanus) and contributes to losses in live holding estimated to be 10-15% under outbreak conditions.The prevalence and impact of this ciliate on wild stocks is unknown.The mechanism of pathogenesis is unknown and having EST data from ciliates recently infecting lobster and comparing to EST data from sea water starved ciliates should provide us with information on modes of infection and pathogenesis.
Additional citations and references	Greenwood SJ, et al..2005.Case report: outbreak of bumper car disease caused by Anophryoides haemophila in a lobster holding facility in Nova Scotia, Canada. Journal of Aquatic Animal Health, 17:345-352.Ragan et al.1996.The Lobster Parasite Anophryoides haemophila (Scuticociliatida: Orchitophryidae): Nuclear 18S rDNA Sequence, Phylogeny and Detection Using Oligonucleotide Primers.Journal of Eukaryotic Microbiology,43:341-346.
Primary citation for organism's characterization, if available	Cawthorn RJ,et al.1996. Description of Anophryoides haemophila n. sp. (Scuticociliatida: Orchitophryidae), a pathogen of American lobsters Homarus americanus. Diseases of Aquatic Organisms 24:143-148.
Latitude	43.51667
Longitude	-65.6
Temperature (ºC)	5
Collection date	01-MAR-04
Collection time	12:30 PM
Sample collection site	Atlantic_Ocean
Other collection site info	The ciliate was isolated from infected lobsters at a lobster holding facility (cordinates above) in Barrington Passage Nova scotia Canada .
Sample material (e.g. "seawater," "sediment," etc.)	lobster hemolymph containing the cliate was cultured in the media listed above. Ciliate was isolated from media for further morphological and molecular characterization.
Habitat description	coldwater holding facility
Other environmental metadata available	Experimental conditions used to isolate and grow cells was as follows, hemolymph (~100 ?L) from moribund lobster was placed into 10 mL modified marine axenic medium (ATCC 1651 MA medium - Messick and Small 1996) composed of artificial sea water supplemented with vitamins (RPMI 1640 vitamins solution, Sigma-Aldrich Ltd., Oakville, ON, Canada), 10 % fetal bovine serum and penicillin / streptomycin (100 U/ mL, 100 ug/mL) at 4 oC for 24 h.This allowed sufficient time for contaminating cell debris to settle and for intact lobster hemocytes to attach to the plasticware.The Anophryoides haemophila isolate Nova Scotia 2004 was then transferred to fresh media and axenic clonal cultures were established (Greenwood et al. 2005).
Other environmental metadata available	Experimental conditions used to isolate and grow cells was as follows, hemolymph (~100 ?L) from moribund lobster was placed into 10 mL modified marine axenic medium (ATCC 1651 MA medium - Messick and Small 1996) composed of artificial sea water supplemented with vitamins (RPMI 1640 vitamins solution, Sigma-Aldrich Ltd., Oakville, ON, Canada), 10 % fetal bovine serum and penicillin / streptomycin (100 U/ mL, 100 ug/mL) at 4 oC for 24 h.This allowed sufficient time for contaminating cell debris to settle and for intact lobster hemocytes to attach to the plasticware.The Anophryoides haemophila isolate Nova Scotia 2004 was then transferred to fresh media and axenic clonal cultures were established (Greenwood et al. 2005).
Habitat	marine habitat
Country	CANADA
Experimental Data
Date of experiment	03-MAY-11
Growth medium	ATCC 1651 MA medium - Messick and Small 1996 made with Instant ocean
Modifications to growth medium	No L-?- phosphatidylcholine was added to the media
Temperature (ºC)	4
Salinty (psu)	30
pH	8
Investigation type	Eukaryotes
Other experimental metadata available	Ciliates were injected into a lobster at 1x10^5 in 3% NaCl. The lobster was held at 4C until moribund (4 weeks). Hemolymph containing ciliates was removed (1 ml aliquots) and cultured in ATCC1651 MA medium overnight to allow contaminating hemocytes and debris to adhere to the plastic containers. Ciliates (6,000 cells/ml) were transferred to fresh media and grown to sufficient density (109, 000 cells/ml). Cells were then washed with fresh artificial seawater (FASW) and resuspended in FASW at a density of 12,500 cells/ml and starved for 11 days. RNA was extracted by RNeasy kit with DNAse1 treatment and quantified by Nanodrop and quality assessed by Bioanlyzer.