Wera 2008 Uw

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UW WERA report

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Wera 2008 Uw

  1. 1. Overview of Shellfish Activities at the University of Washington Updates from Carolyn Friedman and Steven Roberts WERA 2008 April 06, 2008
  2. 2. 1) Population Genetics (and other) – Abalone – Geoduck 2) Quantitative Molecular Diagnostics – OsHV - Oyster Herpes Virus – RLP - Withering Syndrome – QPX and related taxa 3) Physiology [Gene Discovery Efforts] – Pacific Oyster – Abalone – Hard Clams (& QPX) and Manila Clams
  3. 3. • Make changes to abalone slides indicating that: – Pilot scale outplant was a success – Adult aggregation pilot – Further outplanting work (conventional vs habitat enriched) – Disease/ Genetic screening
  4. 4. COI OR2 Flat OR WA 1901 OR Flat 9 Abalone OR Flat 8 OR Flat 7 OR Flat 4 33 WA 1903 OR Flat 6 WEIRDO/ FLAT OR Flat 3 52 WA 1900 Hw a1 flat CA OR Flat 2 Lysin OR Flat 10 62 73 WA 1916 16 OR Flat 1 70 WA 1906 10 WA 1916 WA 1924 22 CA Flat 21 Orf1 flat Or 890 White Catalina WA 1918 68 11 Flat/ Divergent WA 890 White Catalina (2) 57 WA 1901 62 893 White Catalina WHITE 17 CA Flat 22 892 White Catalina 70 891 White Catalina CA Flat VD110 100 70 CA 1 Pinto CA WA 1903 67 45 WA 1904 WA 1905 AKK16 Pinto Ketchikan Has 4 99 Has2 assim SantaBarbara 55 WA 1920 Has 3 PINTO/ THREADED Has3 assim SantaBarbara Has 6 CA 2 Pinto CA 63 99 WA 1904 AKK17 Pinto Ketchikan 69 Has1 assim SantaBarbara WA 1902 Pinto/ Threaded WA 1898 Ak S 15 66 My22 Red Monterey RED Has 2 100 SMI 143 Red SanMiguel Has 7 UW0665 Black Carmel BLACK 100 UW0676 Black SNI CA Pinto 1 62132 Pink SanClemente Has 5 PINK 100 Mex1 Pink Mex Pink lysin INTRON 2(Nathan) Pink Green14 Green SanDiego GREEN 100 LJ7 Green LaJolla 0.005 0.01
  5. 5. Hap F (1) PC 1 Hap A (9) Hap B (8) Hap I (1) 1 1 PA, PC, PW PW, TC TC Hap E (2) 2 PW, PC 1 1 1 Hap C (3) Hap G (1) Hap D (2) 1 PC, PW TC PW 1 2 Hap J (1) Hap H (1) PA PA 5 Lysin Intron 2 404 bp 1 Haplotype Network Hap J (1) Pinto/Threaded Clade DW? 19 Flat/Div.
  6. 6. Abalone http://www.vetigastropoda.com Pinto www.gastropods.com www.gastropods.com All divergent pintos Flat from ONE location Biological significance of observed differences—fertilization studies beginning
  7. 7. Other abalone updates • Pilot outplant successful! (~20% survival after 1yr, > survival in larger size class) • Initiated adult aggregation study • Just funded to begin a larger scale outplant, with hatchery rearing in conventional & habitat-enriched tanks
  8. 8. Geoduck: Effects of Geoduck Aquaculture on Wild Populations? • Are cultured geoduck spawning? • If yes, what is the relative reproductive success of cultured geoducks? • Are cultured geoduck genetically distinct from wild geoducks? • Triploidy?
  9. 9. Maturation and Spawning rates in intertidal geoducks 90 80 70 60 50 MATURE (%) 40 SPAWNED (%) 30 20 10 0 2 year olds 5 year olds
  10. 10. 1) Population Genetics – Abalone – Geoduck 2) Quantitative Molecular Diagnostics – Oyster Herpes Virus – RLP - Withering Syndrome – QPX and related taxa 3) Physiology [Gene Discovery Efforts] – Oyster – Abalone – Hard Clams (& QPX) and Manilla Clams
  11. 11. OsHV QPCR Comparison • QPCR tests developed independently – A (UW) – B, C, GP, and DNA polymerase gene (IFREMER) • Goal: compare sensitivity of QPCR tests – same standard curve (purified OsHV genome) – samples from USA, China, Japan and France – samples with limited OsHV DNA – similar recipes (SYBR) on same QPCR platform (Stratagene MX 3000) C A GP B C X TRL IRL IRS TRS UL US To learn more come to this groovy talk by: Colleen Burge….here at NSA
  12. 12. Oyster Herpes Virus (OsHV): Breeding for Resistance • Losses of seed oysters associated with OsHV in 13 of the past 15 yr in Tomales Bay, CA • Creating potentially OsHV resistant oyster families for outplant in Tomales Bay in 2008 • Survivors of 2003 mortality event, including high and low performing stocks • Families will be monitored for OsHV (QPCR) and other diseases (histology), survival, and growth monthly as compared to a typical hatchery stock
  13. 13. Pacific oyster development and survival Goal: Develop and implement tools to study factors involved in Pacific oyster development and survival – Immune response (including anti-viral genes), growth, and environmental stress (both natural and anthropogenic) – A bioinformatic approach has been used to chose genes and/or Expressed Sequence Tags (ESTs) – Gene specific primers designed for Reverse Transcriptase (RT) QPCR to examine differences in gene expression – Compare pacific oyster tissues collected from Tomales Bay in a year without disease pressure (2007) to Pacific oyster larvae and seed challenged with OsHV Funded by: National Sea Grant Oyster Disease Research Program Work by: Colleen Burge, Carolyn Friedman, and Steven Roberts
  14. 14. Withering Syndrome (WS) QPCR- Diagnostic Validation and Analytical Sensitivity Goal: Compare QPCR quantification to traditional histological indices Preliminary Results: 1) Bacterial loads in QPCR are representative of semi- quantitative histological indices Fluorescence 2) QPCR can detect as few Intial Copy #’s 30 M 3M as three copies of target 300K 30K 3K 300 30 3 DNA 5 10 15 20 25 30 35 40 Cycle Number
  15. 15. WS: Black Abalone Disease Resistance WS Resistance Trial Mean Mortality (No.) 35.00 30.00 p<0.01 • Goal: Assess whether 25.00 San Nicolas Island progeny of WS survivors are 20.00 15.00 Carmel more resistant than are 10.00 5.00 naïve or non-selected 0.00 populations 2/4/2007 4/4/2007 6/4/2007 8/4/2007 10/4/2006 12/4/2006 10/4/2007 12/4/2007 • Method: Challenge two groups Time (Date) of black abalone to WS-RLO: Group 1: progeny of WS Termination Sampling Histology survivors (San Nicolas Island) 3 Relative Response p<0.05 p>0.05 p<0.05 Group 2: recently exposed to 2.5 2 WS-RLO but not active 1.5 Carmel San Nicolas Island disease (ie no WS) (Carmel) 1 0.5 0 META PE TOTAL DG TOTAL BACT BACT Category
  16. 16. RLP loads of Black ab Morts via QPCR 35000 Carmel DNA copies/ng Genomic DNA 30000 25000 20000 SNI 15000 10000 5000 0 Black Ab RLP loads via QPCR 30000 DNA copies/ng Genomic DNA 25000 Carmel late 20000 SNI late 15000 Carmel early 10000 5000 SNI early 0
  17. 17. WS: Two Novel bacteria infecting abalone New RLO WS RLO Stippled RLO
  18. 18. Analyses of novel bacteria infecting abalone • Antimicrobial Injection study: Differential susceptibility of the WS-RLO and two new bacteria to three antimicrobial drugs Anti- Stippled New microbial WS RLO RLO Bacterium Chloramphenicol R R S Clarithromycin R S R Difloxacin R S S • BLAST analysis of sequences from cloned amplicon produced using universal eubacterial primers and DNA from heavily infected tissue (more new bacteria than WS-RLOs in the tissue) – WS-RLO identified in 5/96 clones – No other known rickettsia-like bacteria amplified – Further examination of amplified bacteria underway
  19. 19. New RLO cross reacts with WS- RLO ISH probes, while Stippled RLO does not cross react
  20. 20. QPX & more Maille Lyons and Chris Dungan Wed 11:15 Drew Hall Cornell University
  21. 21. • Population Genetics – Abalone – Geoduck • Quantitative Molecular Diagnostics – QPCR for Shellfish Larvae – Oyster Herpes Virus – RLP - Withering Syndrome – QPX and related taxa • Physiology [Gene Discovery Efforts] – Oyster – Abalone – Hard Clams (& QPX) and Manilla Clams
  22. 22. Oyster Immune Mon 1:45
  23. 23. Market size
  24. 24. High Mobility Group Protein
  25. 25. Markers ?
  26. 26. Summary - Field Trial • Offspring of survivors of heavy disease pressure are more tolerant to disease • Mechanisms involved in host responses to P. marinus include proteases and apoptosis • Careful selection of broodstock (potentially aided by markers) can benefit aquaculture and conservation efforts
  27. 27. Abalone, clams and clams Mon 9:30 Wed 11:15

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