Mussels feeding on sea lice IMTA systems and disease Risk Increase Infectious pancreatic necrosis virus (IPNV) www.veths.no/.../ Research/Aquatic-medicine/ Virology Journal 2007, 4:13 Infectious salmon anemia virus (ISAV) Vibrio anguillarum O2b VIbrio Plate.tif Image10cut.JPG Lepeoptherius salmonis Decrease Thanks Funding crest-top.jpg nracLogo.gif Bricknell Lab Dr. Sarah Barker Dr. Sally Dixon-Molloy Mike Pietrak Jennifer Fortier Erin Switzer Hillary Scannell Neil Greenberg Chris Roy Bouchard Lab Emily Thomas Sarah Turner Debbie Bouchard Integrated Multitrophic Aquaculture. How to Manage Diseases in an Artificial Ecosystem Dr. Ian Bricknell, Director Aquaculture Research Institute University of Maine, Orono ARI Logo transparent.tif UM.png Ancient Aquaculture First recorded in hieroglyphs from the middle kingdom 1786-2052 BC Earth pond system Broodstock animals put in at the end of the Nile flood period Harvested just before next floods Ancient Aquaculture (Egyptian middle kingdon) Dry season Nile Dig earth ponds on the flood plane Wet season:- Nile floods and fills ponds Fish left in flooded ponds Additional animals added Harvest at end of dry season waste also used as a crop fertiliser Integrated Multitrophic Aquaculture (IMTA) Principals The waste products from one crop are used to grow secondary crops. For example cow manure is used to grow corn and other fodder crops to feed the main crop In aquaculture the organic particles are used to grow shellfish The inorganic wastes (nitrogen and phosphorus) are used to grow sea weeds IMTA is not a new idea Hawaii started IMTA in the 1300’s Captain Cook recorded over 364 active sites in the 1778 Production estimated at 900 metric tons in 1778 Upland water diverted to grow Taro lo’i Swift streams carry water from Taro fields to Ohi’a koa & kukui plantations ‘auwai ditches & pools used for freshwater fish culture The flood plane is used to grow more Taro lo’i This system drains into a rock wall fish pond for finish aquaculture. The nutrients this water carries causes plankton to bloom and provides food for the fish http://fishpondfever.wordpress.com/about/ IMTA Can form part of a terrestrial integrated farming practice (Asian model). Farm builds are built over fish ponds Top floor is usually Poultry Bottom floor is usually pigs Waste from the foul is fed to the pigs Pig waste is added to the fish ponds (as are other organic waste products) This eutrophicates the water causing plankton to bloom and aquatic macrophytes to grow rapidly This feeds the fish IMTA Can form part of a terrestrial integrated farming practice (Asian model). Chickens kept above pigs Pigs kept above fish Fish kept below pigs Pigs feed on chicken waste Pigs waste eutrophicates water and provides fish nutrition Inputs: 340 tonnes manure/farm/household waste Outputs: 8 tonnes of fish 10cm enriched sediment 20 tonnes of Grass 10 tonnes feed Bio-economics Pond polyculture Asian carp system Grass Carp – Herbivorous, feeds on macro-vegetation Silver Carp – Feeds on phytoplankton Bighead Carp – Feeds on macroplankton Black Carp – feeds on snails and other mollusks Mud carp – Feeds primarily on detritus Depth So why isn’t IMTA widely practiced in developed countries today? Western fish farming restarted in the 19th century typically farming brown or rainbow trout In the 1960’s Atlantic salmon life cycle was broken Marine Harvest patented the technology This was based on a single species fish farm It set the model for fish farming in the western world for 40 years Pro and Cons of single species fish farming One environments to manage One feed regime One health regime Vaccines against disease for 1 species Chemotherapeutics suitable for only one species Unified harvest Simple staff training Dedicated equipment Multiple environments to manage Several feed regimes Multiple health regimes Treatment for the main crop may adversely impact a secondary crop Secondary crop may act as a reservoir for pathogens Asynchronous harvests Complex staff training Multiuse equipment 44993-R1-08-9.jpg Farmed Fish vibrio 1.tif vibrio 1.tif Pathogen Pathogen Cages CW.jpg Environment So Why IMTA? Mitigates the carbon footprint of aquaculture Provides site diversification Helps farms in time of economic hardship if the main crop declines in value Provides more employment Highly efficient use of water http://www.proaqua.com/aquaponi cs Integrated Multi-Trophic Aquaculture Typical Maine IMTA system Fed Aquaculture Extractive Aquaculture Mussels Kelp (Sweet Kelp) IMTA benefits PB280145 Shawn Robinson, DFO Diversification of products Environmental sustainability Economic sustainability Disease Risk? Mussels as filter feeders may bio-accumulate pathogens Integrated Multi-Trophic Aquaculture Typical Maine IMTA system Fed Aquaculture Extractive Aquaculture Mussels Kelp (Sweet Kelp) IMTA benefits PB280145 Shawn Robinson, DFO Diversification of products Environmental sustainability Economic sustainability Disease Risk? Mussels as filter feeders may bio-accumulate pathogens The question we had is do IMTA Ecosystems increase disease risk? egg Periwinkle Cercaria X Periwinkles live between 0-100ft. On rocky surfaces, so move fish pens into deeper water or into sandy habitats Cryptocotyle life cycle IMTA benefits PB280145 Shawn Robinson, DFO Diversification of products Environmental sustainability Economic sustainability Disease Risk? Mussels as filter feeders may bio-accumulate pathogens The question we had is do IMTA Ecosystems increase disease risk? egg Periwinkle Cercaria X Periwinkles live between 0-100ft. On rocky surfaces, so move fish pens into deeper water or into sandy habitats Cryptocotyle life cycle Possible Pathways vibrio 1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif Current flounder_summer.png flounder_summer.png herring.tif herring.tif herring.tif pollock.png 44993-R1-08-9.jpg Farmed Fish vibrio 1.tif vibrio 1.tif Pathogen Pathogen ? mussel_close.jpg mussel_close.jpg ? Cages CW.jpg Environment Research question Fate of pathogen in system will be highly dependent upon pathogen physiology Mussels = biological filters of pathogens ( disease risk) OR Mussels = vectors for pathogens ( disease risk) ? Infectious pancreatic necrosis virus (IPNV) www.veths.no/.../ Research/Aquatic-medicine/ Virology Journal 2007, 4:13 Infectious salmon anemia virus (ISAV) www.uri.edu/.../ dnelson/interests_02.html Vibrio anguillarum O2b Photo: Duncan Colquhoun Francisella philomiragia Lepeophtheirus salmonis Photo: Mike Pietrak The approach Investigate interactions of each pathogen with mussels and Atlantic salmon. Fate of each pathogen in mussels + ? http://images.google.com/images /isr_c.gif Field investigation using sentinel mussels at marine grow-out sites http://www.merassessment.com/default%20images/aqua_fishfarm_270x195.jpg www.merassessment.com/Level_2_pages/clients_l... Remove frame Ecology of pathogen in fish/mussel systems compared to fish only systems ? Fate of ISAV and IPNV in mussels ISAV 104 TCID50/ml Digestive gland Virus Detection Tissue culture infectious dose (TCID50) qRT-PCR Enveloped 120 nm Fate of ISAV in mussels ISAV genome present in tissues. NO viable ISAV was detected in mussel tissues. All tissue-culture assays negative for ISAV in mussel tissues Relative abundance of ISAV segment 8 in mussel digestive glands after exposure to ISAV 2- Viruses Smaller than optimal mussel particle  Less efficient at filtering ISAV belongs to this group Less environmentally hardy Susceptible to lipid envelope being removed Mussels digest the lipid envelope Enveloped Viruses Fate of ISAV in mussels ISAV genome present in tissues. NO viable ISAV was detected in mussel tissues. All tissue-culture assays negative for ISAV in mussel tissues Relative abundance of ISAV segment 8 in mussel digestive glands after exposure to ISAV 2- Viruses Smaller than optimal mussel particle  Less efficient at filtering ISAV belongs to this group Less environmentally hardy Susceptible to lipid envelope being removed Mussels digest the lipid envelope Enveloped Viruses ISAV transmission trial No mortality in any group No test fish ISAv +ve Mussels ISAv Genome +ve ISAv not viable by TC Or shown to be replicating in mussels ISAV infected salmon Water from infected salmon Passes over mussel tank ISAV free salmon live In mussel treated water for 28 Days Conclusions ISAV was not transmitted to Atlantic salmon after processing by mussels Inactivation mechanism is believed to be due to the mussels stripping the lipid layer of enveloped virus Viruses Smaller than optimal mussel particle  Less efficient at filtering IPNV is a non-enveloped virus Environmentally hardy Transmission can occur via mussel Feces May replicate in mussels? Non-Enveloped Viruses http://www.reoviridae.org/dsRNA_virus_proteins/Aquabirnavirus-IPNV.htm Aquareovirus particle.gif Fate of IPNV in mussels Viable IPNV detected in tissues. Confirmed by qPCR. Non-enveloped 60 nm IPNV shed in mussel feces Viable IPNV shed in mussel feces Random Block Design Cohabitation IPNV challenge Cohabitation IPNV challenge Sentinel 6 Fish/tank sampled weekly Do IPNV-loaded mussels transmit IPNV to Atlantic salmon? 8 Day Exposure 106 TCID50 ml-1 IPNV MEM Log TCID50 g-1 5.2 ± 0.16 18 Day Exposure Log TCID50 g-1 3.1 ± 0.04 No virus detected IPNV positive fish – culture Cohabitation IPNV challenge Sentinel IPNV negative IPNV + IPNV + Mortalities Mortalities   Rep A1 Rep B1 Rep A2 Rep B2 I.p injected 12/12 12/12 0 0 Cohabitant 1/12 1/12 0 0 IPNV + IPNV + IPNV +   Rep 1 Rep 2 Rep 3 System A 1/24 2/24 0/24 System B 1/24 1/24 1/24 ISAV QPCR – Mussels remove ISAV from water. Culture – No viable ISAV detected in mussel tissues. Salmon i.p. injected w/tissue homogenates from ISAV-loaded mussels ≠ No disease Do mussels on IMTA farms increase or decrease viral disease risk? IPNV QPCR + Culture - Mussels accumulate IPNV in tissues. Culture – Viable IPNV in mussel tissues and feces after depuration. IPNV transmitted to fish cohabitating with IPNV-loaded mussels Bacteria Environmentally hardy Can bio-accumulate Transmission can occur via mussel Feces Opportunistic Bacteria VIbrio Plate.jpg ISAV QPCR – Mussels remove ISAV from water. Culture – No viable ISAV detected in mussel tissues. Salmon i.p. injected w/tissue homogenates from ISAV-loaded mussels ≠ No disease Do mussels on IMTA farms increase or decrease viral disease risk? IPNV QPCR + Culture - Mussels accumulate IPNV in tissues. Culture – Viable IPNV in mussel tissues and feces after depuration. IPNV transmitted to fish cohabitating with IPNV-loaded mussels Bacteria Environmentally hardy Can bio-accumulate Transmission can occur via mussel Feces Opportunistic Bacteria VIbrio Plate.jpg Persistence of pathogen in mussel digestive gland over time Experiment 1 Fate of Vibrio in mussels Mussels concentrate viable V. anguillarum in their tissues but do they shed viable Vibrio? VIbrio Plate.jpg Transmission to Fish Experiment 1 hr challenge of cod to feces from mussels exposed and unexposed to Vibrio anguillarum 02β cod.tif cod.tif V. anguillarum can infect fish via fecal matter Va Mort Bar graph.png V. anguillarum Transmission Current Sediments ARI.jpg vibrio 1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif altfemale1.tif X ? Lab vs Farm Experimental conditions favor transmission BMPs can minimize transmission risks Disease can be managed with vaccines Parasites Sea lice Complex life cycle Has 3 planktonic stages May be possible to control shedding of the parasite biologically _1030655.JPG Experimental fish sea lice infection trial Sea Lice – Planktonic stages Ideal size range to be filtered (Molloy et al. 2011) Larval stages rendered non-infective Trapping in gill mucus or entering the digestive tract kills them Mussels can remove from water column in a flow – (Shawn Robinson DFO) 2010_11_10_15_45_45.tif 2010_11_10_15_51_57.tif