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Non-Elucidated Mortality Events of Juvenile Hard Clams in a Hatchery System John Aldred East Hampton Town Shellfish Hatchery Steven Pitchford NOAA/NMFS Milford Labs Bassem Allam SUNY/Stony Brook ---PAGE BREAK--- East Hampton, NY ---PAGE BREAK--- Field Grow Out Hatchery Nursery ---PAGE BREAK--- Products Oyster Scallop Clam ---PAGE BREAK--- Brood Stock Conditioning • Static water @ 16 C • Water change 3x/week • Tank, feed tank, piping & tubing clorox washed bi- weekly • Continuously drip fed a mix of Thalassiosira weisflogii, Pavlova lutheri, galbana • Six week conditioning period • One batch oysters, three batches clams, several batches scallops ---PAGE BREAK--- Raw Water Receiving Tank Origin of water used throughout hatchery • Incoming water 1 micron bag filtered • Tank brought to temperature by recirculating through heat exchanger • Gravity fed to culture tanks • Above in continuous process • Tank, piping, hoses chlorinated overnight filter bags weekly • Intake pipes freshwater flooded overnight ---PAGE BREAK--- Algae Stock and Flagellate Culture galbana and Pavlova lutheri • Algae room @ 20 C • 1 micron filtered water • Autoclave flasks • Chlorinate/dechlorinate carboys & kalwall tubes • Batch culture to 7 days maximum • Batch feed flagellates from tubes • Glass & plastic ware clorox or acid washed as emptied • Fill and drain piping & tubing chlorinated ---PAGE BREAK--- Diatom Mass Culture Thalassiosira weisflogii or Tetraselmis spp. • Second story under clear roof panels • Chlorinate/dechlorinate 1 micron filtered culture water • Batch culture to 5 days maximum • Batch feed • Oxalic acid wash tanks • Tanks and feed piping & tubing chlorinated overnight ---PAGE BREAK--- Larval and Post Set Culture • 100 gallon larval conicals • 200 gallon set tanks • Cultured @ 24- 27 C • Tanks & tubing clorox washed bi- weekly • UV filter available in tank fill line • Animals leave room @ 1mm ---PAGE BREAK--- Recirculated downwelling (prior to ambient upwelling) • Animals introduced at 26 C, cools to 20 C • Kept here about 2 weeks for additional growth and ‘hardening off’ • Hatchery schedule set to deliver first clam seed to upwelling system when ambient water reliably reaches 15 C (mid May to early June) • Initial mortalities observed in this system in May of 1996, 2001, 2003 ---PAGE BREAK--- History of Problems • 1994 – No growth, subsequent mortality in upwelling system in Montauk hatchery. Suspected oceanic algal makeup. Upwelling system moved to more estuarine location. • 1996 – Sudden mortalities in 1mm clams in hatchery downwelling system. Subsequent unaffected batches succumbed sequentially. Suspected new algae. Algae challenge trials in 1997 did not replicate mortality. • 2001 – Mortalities similar to 1996 in downwelled 1mm clams. Histopathology revealed mycosis as cause of death. Problem somewhat managed by moving to upwelling system. • 2003 – Mortalities similar to 1996 & 2001. Histopathology revealed only unknown chain structures. Problem again managed by early move to upwelling system. ---PAGE BREAK--- 2001- Sections through the Adductor Muscle and Gill Atrophied Adductor Muscle Erosion of the Gill Inke Sunila, CT Bureau of Aquaculture ---PAGE BREAK--- Fungal Hyphae 2001 - Section showing fungal mycelia with thin hyphae Inke Sunila, CT Bureau of Aquaculture ---PAGE BREAK--- Bacterial Counts June 10, 2003 Steven Pitchford NMFS, Milford Labs Source Media Colonies/ml Comments 1. Inlet Sea water TCBS MA 0 3.7 x 103 T=15C 2. Recirculating Sea water system TCBS MA 20 2.3 x104 1u bag filtered, heated to >25C from open conical upstairs (Head Tank) 3. Thalassiosira Open conicals upstairs (Mass Algae Culture Tanks), temperature not taken but >25C 3a. Tank 1 (6/7 batch) TCBS MA 20 6.0 x 105 3b. Tank 2 (6/9 batch) TCBS MA 0 2.7 x 104 3c. Tank 3 (oldest- no date) Tank with thick bottom sludge layer. Two samples taken, one from the surface and the other directly from the sludge layer. 3c(1) Surface TCBS MA 0 2.0 x 105 3c(2) Sludge layer TCBS MA 140 1.2 x 108 Algal Paste TCBS MA 0 >6.7 x103 Spread directly onto plates, no dilutions performed or sample brought back to lab. Bacterial population may be much higher. ---PAGE BREAK--- 2003 – Preserved Clam Samples Fix Date Batch Length (mm) System Location Degree of Infection Final disposition of batch after move to upwellers 5/19 C1 ± 1.0 Dwl + Moderate survival 5/19 C2 ± 1.0 Dwl Little survival 5/19 C2 ± 1.0 Dwl Little survival 5/19 C3 ± 0.3 Set Tanks _ High survival (early move) No survival (late move) 6/04 C4 ± 0.15 Conicals _ Eventually little survival ---PAGE BREAK--- 2003 - Recently set healthy juveniles H&E stain Bassem Allam, SUNY, Stony Brook ---PAGE BREAK--- 2003 - Low magnification of a section from a juvenile batch with low mortality Atrophied adductor muscle A careful look at the surface of the shell shows chain-like structures Bassem Allam, SUNY, Stony Brook ---PAGE BREAK--- Grocott's Methenamine Silver (GMS) stain 2003- In early stages, chain-like structures are seen first on external surface of the shell Shell Chain-like microorganisms Bassem Allam, SUNY, Stony Brook ---PAGE BREAK--- Pallial (mantle) cavity Mantle 2003 - In more advanced stages, chain-like microorganisms are seen inside the pallial cavity (mantle, gills) Bassem Allam, SUNY, Stony Brook ---PAGE BREAK--- …and an erosion of the pallial organs (mantle, gills) is seen… Gill Bassem Allam, SUNY, Stony Brook ---PAGE BREAK--- Focus Area 1 Recirculating receiving water system acting as a microbiological breeding ground Possible strategy: Add in-line UV ---PAGE BREAK--- Focus Area 2 Settled Cells in Mass Algae tank acting as microbiological breeding ground Possible strategy: Avoid stirring tank of taking off feed from tank bottom ---PAGE BREAK--- Focus Area 3 Periodic presence of harmful algae as stress factor (In 2003 Gymnodidium identified as problem source in Noank, CT and ‘dinoflagellate-like cells’ were found in dying juvenile oysters in Peconic Bay) Regular assessments of the algal communities in shellfish growing areas would be useful