Enhancing Stress Resistance of. Final Report on Field Trials and Laboratory Challenges

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Hugo Young
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1 Enhancing Stress Resistance of Cultured Clams Through h Triploidy: id Final Report on Field Trials and Laboratory Challenges John Scarpa, Harbor Branch Oceanographic Shirley Baker, UF, SFRC, Fisheries and Aquatic Sciences Leslie Sturmer, UF Cooperative Extension Service Chuck Adams, UF Department of Food and Resource Economics

2 Hypothesis Mortalities from summer stressors can be reduced by creating sterile clams through triploidy Triploidy = 3 sets of chromosomes

3 Why triploidy? Triploids divert energy from reproduction to storage and growth Used in PNW oyster aquaculture Need for hardier clam strain in SE

4 Specific objectives Create replicate diploid/triploid families Compare growth & survival during grow-out out Compare physiological responses to stress Examine economics

5 Meiotic and Mitotic Stages in Eggs of Mercenaria mercenaria Sperm Metaphase I Early anaphase I Late anaphase I PB 1 PB 2 Pronuclei Syngamy 1 st Cleavage (2-Cell stage)

6 Mean Time to Formation of First Polar Body (PB 1), Second Polar Body (PB 2), and First Cleavage in Hard Clam Eggs 60 Perce entage of cells at each stage PB 1 PB 2 1st cleavage Time (min) post-insemination

7 Flow Fow Cytometry try Polar Body 2 release Polar bodies Flow Cytometry output Male genome

8 Cytological and Flow-Cytometric Data from Triploid Induction Experiments Trial #, treatment 1 Cytological Data (% of fertilized eggs) Prefertilized Pre-PBI Pre- PBII Post- PBII Triploid (%) PBI PBII PBI PBII PBI PBII PBI PBII PBI PBII PBI PBII PBI PBII PBI PBII

9 Juvenile Growth Studies

10 Growth (%) in shell length (A) and live weight (B) of juvenile triploid Mercenaria mercenaria th) GROWTH (% incre ease in Shell Lengt A a b b Diploid a b b PB 1 triploid PB 2 triploid a b b a a a Total TIME (weeks) G R OWTH (% incre ase in live w eig ht) B Diploid a b c PB 1 triploid PB 2 triploid a b c a b b a a b Total TIME (weeks)

11 GROWTH (% increa ase in live weight) Growth (% live wt) of juvenile triploid Mercenaria mercenaria in experiment two K M O All FAMILY Diploid Triploid

12 Survival under stress 45 mm SL Temperature: 90 C Salinity: 10, 25, 40 ppt Oxygen: Normoxia or hypoxia Mortality

13 Survival 10 and 40 ppt 10 ppt 40 ppt tive Survival % Cumulat Diploid, 10ppt, Normoxia Triploid, 10ppt, Normoxia Diploid, 10ppt, Hypoxia Triploid, 10ppt, Hypoxia tive Survival % Cumulat Diploid, 40ppt, Normoxia Triploid, 40ppt, Normoxia Diploid, 40ppt, Hypoxia Triploid, 40ppt, Hypoxia Time (Days) Time (Days) At li it t t i l id l h At salinity extremes, triploid clams have no advantage over diploid clams

14 Survival 25 ppt 100 % Cumulativ ve Surviva l Diploid, 25ppt, Normoxia Triploid, 25ppt, Normoxia Diploid, id 25ppt, Hypoxia Triploid, 25ppt, Hypoxia Time (Days) Triploidy may increase survival in hypoxia

15 Physiological response to stress Oxygen uptake rates 50 mm SL 25 ppt or 15ppt 68, 77, 80, 86, and 90 F

16 Oxygen uptake rate temperature Log oxygen con nsumption n (ug/g/h) Diploid Triploid Metabolic rate increases with temperature, but not above 80 F Triploid clams have no advantage over diploids Temperature ( F)

17 Oxygen uptake rate salinity consumpt tion (ug/g/ /h) Log oxygen 25 ppt 15 ppt Triploid clams have lower metabolic rate at 15 ppt than at 25 ppt (80 F) Triploid clams may use less energy during stress

18 Comparison of Survival and Hsp70 Synthesis in Triploid and Diploid hard clams under Normal and Heat Shock Conditions. (Sampling times: 0T (day 10), 4 hr, d1, d5 or d7) 0 Time Diploids 10 days Start of Heat shock 5-7 days End of experiment Acclimation Control 25 0C 25 C 25 C 25 C Acclimation 35 C Heat Shock 35 C Triploids Acclimation 25 C 25 C Control 25 C 25 C Acclimation 35 C Heat Shock 35 C

19 Experimental System (1) Acclimation Unit : Sump connected to a 25±1 o C. (2) Cold Treatment Unit: Sump connected to a 25±1 o C. (3) Hot Treatment Unit: Sump with titanium 35±1 o C. (1) (2) (3)

20 Hs sp70 (RU µg TSB -1 ) Hsp70 and Survival (± SD) in Triploid and Diploid Hard Clams (SL=31.6 ± 6.6) Following Severe Heat Shock (25-35 o C) T0 4h D1 D7 Time after heat shock 3N 2N Surviv val (%) Time (days) after heat shock 3N-M 2N-N

21 Survival (± SD) of hard clams (SL= 44.0± 3.3) exposed to severe heat shock (25-35 o C) was influenced by genetic background represented by higher initial Hsp70 concentration (P= 0.042), 042) than by ploidy (P= 0.184). 3N-K 2N-L 3N-M 2N-N ) Survival (%) T 0 Day 1 Day 2 Time following heat shock ) Survival (%) T 0 Day 1 Day 2 Time following heat shock Survival (%) 3N-O 2N-P T 0 Day 1 Day 2 Time following heat shock Hsp70 ( RU µg TS SP -1 ) A A B a a a a a a K & L M & N O & P Family 3N 2N

22 GROW-OUT OUT Leslie Sturmer Clam samples

23 Hurricane Charley

24 Mean values of different parameters measured for PB2 triploid clams cultured in Cedar Key Diploids Triploids T-test N Mean SD N Mean SD Signif. Shell Length ± ± Shell Width ± ± Live Weight ± ± Dry Meat Weight ± ± Condition Index ± ± Four hurricanes hit Florida in 2004 and destroyed 80% of all clams planted for the study. Data presented is from only one group of clams cultured in Cedar Key and sampled in December Triploid clams were estimated t at 42-70% before the hurricanes, but only 29% after. Triploid id clams were significantly ifi smaller for all parameters measured except condition index. Histological analysis indicated 50% of diploid clams had spawned, whereas 100% of triploids had no mature gonad.

25 Cedar Key Grow Out 2 (PBII triploids) Ploidy Length Weight Cond. Survival (mm) (g) Index (%) 2N N Charlotte Harbor Ploidy Length Weight Cond. Survival (mm) (g) Index (%) 2N N

26 Economics (Cost Categories) Broodstock Conditioning: 2x # of clams (capital investment, not calculated) Spawning: increase area?, cost? Chemical Treatment: CB/DMSO ~$20.45/5M eggs (= $ /1K 1mm seed) Chemical Waste Disposal: ~$71.20/5M eggs (= $ /1K 1mm seed) Triploidy id Verification: $100/5M eggs (= $ /1K 1mm seed) Larvae Culture: no sign. expense anticipated Setting: no sign. expense anticipated TOTAL = $ /1K 1mm seed or ~5-10% incr.

27 SUMMARY Produced triploid clams Growth of triploids in lab was lower Growth of triploids in field was similar/lower Survival/stress resistance of triploids in lab exhibited mixed results (Hsp70 selection) Survival of triploids in field was lower CI of triploids in field was similar/greater Cost of producing triploids is minimal No apparent advantage of triploid clams for Florida culturists t

28 THANKS to Florida Sea Grant (R/LR-A-39) and USDA support Many, many people who assisted in collecting, caring, experimenting, and analyzing clams: Tony Heeb (Cutthroat Clams) Roy Kibbe (Kibbe Clams) Dan Leonard (Bull Bay Clams) Chris Taiani (Cedar Key) Terry Lange (Ft. Pierce) Eric Cassiano, Elise Hoover, Kerry Weber (UF) Kyrstal Baird, Fred Prahl, Chris Withstanley (HBOI) Eman El-Wazzan (FIT) and those we have missed Questions?

Triploid-tetraploid Technology for Oyster Aquaculture Industry

Triploid-tetraploid Technology for Oyster Aquaculture Industry Huiping Yang University of Florida 7922 NW 71st Street, Gainesville, FL 32653 Phone: 352-294-0671, Email: huipingyang@ufl.edu 3618 Coastal