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Spawning and Larval Rearing of Geoduck (Panopea Generosa)

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Spawning and Larval Rearing of Geoduck (Panopea Generosa) Spawning and Larval Rearing of Geoduck (Panopea generosa) Jesse Ronquillo, PhD North Island College Campbell River, BC Panopea generosa Gould, 1850 (Pacific Geoduck Clam) Scientific classification Kingdom: Animalia Phylum: Mollusca Class: Bivalvia (Pelecypoda) Order: Myoida Family: Hiatellidae • World’s largest burrowing clam. • Weighs 0.5-1.5 kg at maturity. Genus: Panopea • Maximum weight: 7.15 kgs. Species • Live to over 140 years and reach a Panopea generosa Gould, 1850 maximum shell length of 23 cm. Pacific Geoduck Clam • Oldest geoduck clam aged from British Columbia is 168 years old Pacific Geoduck Clam Geographic Distribution Pacific Geoduck Clam • Geoduck is the most important commercial shellfish species in British Columbia. • Commercial production is limited by the reliable supply of high quality hatchery- produced juveniles. • Developing viable and sustainable culture protocols for this species will provide the industry with the needed seedstock to expand production. Objectives • Determine the best algal diet for larval geoduck; • Develop inexpensive system for efficient and continuous culture of microalgae for feeding geoduck seedstock. • Manipulate geoduck broodstock spawning; • Design and construct of a prototype closed- culture system for controlled geoduck spawning; • Fabricate an inexpensive seawater filtration system for geoduck hatchery operation; Broodstock Disease Collection & Diagnosis Maintenance Genomics & DNA Markers Spawning/ Algal Incubation Production Larval Rearing Juvenile Rearing Nutritional Statistical Analysis Analysis Schematic Flow Diagram of Activities PUFA – Polyunsaturated Fatty Acids LA (Linoleic acid, 18:2n-6) ALA (Alpha-linolenic acid, 18:3n-3) AA (Arachidonic Acid, 20:4n-6) EPA (Eicosapentaenoic Acid, 20:5n-3) DHA (Docosahexaeoic Acid, 22:6n-3) Modified from: http://en.wikipedia.org/wiki/Fatty_acid PUFAs EPA & DHA are structural component of brain, eyes, heart tissues and all cell membranes. http://kvhs.nbed.nb.ca/gallant/biology/biology.html Algal Production of HUFA PUFAs Thalassiosira weissfloggii http://www.nies.go.jp/biology/mcc/class/Te traselmis.html Microalgae Thalassiosira weissfloggii Chaetoceros muelleri Nannochloropsis oculata (ACTIN) (CHGRA) (NANNO) Isochrysis galbana (T. ISO) Pavlova lutheri (PAV) Tetraselmis striata (TETRA) Agar Plate Isolation 24-hole plate 100 mL flask 7 L glass container Carboy 1 L flask Algal Scaling Up Culture Algal Production • Algal inocula were procured from UBC and USA. • f/2 medium was used to enrich algal culture. • Microalgae were scaled up from 5 mL test tubes up to 500 L capacity tanks Algal Production Lab 40 algal species www.nsac.ca/pas/aqua Lipid Analysis Summary • Algal Preparation •Fatty acid extracted using MeOH and chloroform •Phases separated using 0.9% potassium chloride •Upper layer filtered through Na2SO4 then evaporated •Methylated by adding Hilditch reagent and heated. •Phase separated via hexane & deionized water. •Filtered upper layer through Na2SO4 •Evaporated, weighed, and run through Gas Chromatography EPA, DHA and AA of Experimental Algal Species Species EPA DHA AA (% DW) (% DW) (% DW) Chaetoceros muelleri 7.00 1.21 4.70 Isochrysis galbana 1.13 9.95 0.11 Tetraselmis striata 13.82 0.21 8.72 Thalassiosira 14.42 3.04 0.29 weissfloggii Nannochloropsis oculata 12.60 0.23 15.65 Pavlova lutheri 13.81 9.65 0.41 PUFA Profile of Algae Treatment ΣEPA Σ DHA Σ AA Σ (EPA+DHA) Σ (DHA+AA) Σ (EPA+DHA+AA) (% DW) (% DW) (% DW) (% DW) (% DW) (% DW) Chagra + 8.13b 11.16a 4.81c 19.29c 15.97b 24.1c T.Iso Tetra + 28.24a 3.25b 9.01b 31.49b 12.26c 40.5b Actin Nanno + 26.41a 9.88a 16.41 36.29a 26.29a 52.7a a Pav (P<0.001***). Microalgal Nutrition Research (In-House Research) Microalgae Chaetoceros muelleri (CHGRA) Thalassiosira weissfloggii (ACTIN) Nannochloropsis oculata (NANNO) Isochrysis galbana (T. ISO) Pavlova lutheri (PAV) Tetraselmis striata (TETRA) Level of PUFA in Each Treatment. 11 a 10 18 9 a 16 a b 14 8 a a 12 b 7 a a b AA 10 a,b a b c 6 b b a EPA (g) Weight 8 b.c b 5 a,b DHA c b,c 6 b a a b c 4 a c Level of PUFAs (%) Level of PUFAs 4 a 2 3 0 2 Chgra+ Iso Nanno+ Pav Tetra+Actin 0 1 2 3 4 5 6 7 Week Oyster Treatment Weight t.iso/chg Weight Nan/Pav Weight Tet/Act (P<0.001). Weekly average weight of oyster juveniles in each diet treatment (P<0.001) Ronquillo, JR, Fraser J, McConkey, AJ. 2012. Effect of mixed microalgal diets on growth and polyunsaturated fatty acid profile of European oyster (Ostrea edulis) juveniles. Aquaculture 360-361: .64-68. Microalgal Study CONCLUSION The best algal diet in shellfish larval rearing was a mixture of Nannochloropsis oculata and Pavlova lutheri (Nanno+Pav). Tetraselmis striata and Thalassiosira weissfloggii (Tetra+Actin) diet has the lowest level of total DHA+AA among treatments; thus, resulted poor growth. Nannochloropsis oculata and Pavlova lutheri (Nanno+Pav) diet has the highest level of omega-3 PUFAs among treatments which made growth faster (P<0.0001***) and higher bioaccumulation of PUFAs in the soft tissues. Geoduck Broodstock Collection & Transport • Geoduck broodstock were collected by diving from Vancouver Island by BCPOL • Broodstock were transported by boat to the facilities at DFO West Vancouver Laboratory and at UBC Faculty of Science. Transfer and Cleaning of Broodstock Gonadal Maturation Analysis Microscopic Analysis of Gonad Smear Geoduck Spawning Geoduck Spawning Spawning at UBC Faculty of Science Lab Spawning at DFO West Vancouver Lab Embryonic Development Newly Spawned Egg and Sperm Newly Fertilized Egg Embryonic Development First Polar Body and Two-Cell Stage Development of Daughter Cell Embryonic Development Four-Cell Stage Eight-Cell Stage Embryonic Development 16-Cell Stage 32-Cell Stage Post-Embryonic Development D-shape Veliger, 7 days Umbone Stage, 15 days Post-Larval Stages 30 days old, 1.2 mm 35 days old, 3 mm Early Settling Stage 50 days old geoduck juveniles (7-12 mm) Newly spawned Fertilization Stage 2-Cell Stage 4-Cell Stage 8-Cell Stage egg and sperm 16-Cell Stage Juvenile 32-Cell Stage 50 days old geoduck juveniles (7-12 mm) Pediveliger Veliger Comparative Rearing (85 Days Old Juveniles) Culturing Without Substrate Culturing in Sandy Substrate Burrowing Behaviour Early Juvenile Feeding Behaviour Culturing Without Substrate Culturing in Sandy Substrate Early Juvenile Behaviour Disease Analysis Conclusions • The preliminary research outcomes include: – (1) Design and construction of a prototype closed-culture system for controlled geoduck spawning; – (2) Fabrication of an elegant yet inexpensive seawater filtration system; – (3) Successful induction of geoduck broodstock spawning based on environmental manipulation among desiccation, temperature shifts, UV-filtered water stimulation, microalgal addition, and various combinations of the above; and – (4) Successful development of inexpensive system for efficient and continuous culture of microalgae for feeding geoduck seedstock. ACKNOWLEDGEMENT • BC Pacific Oysters Ltd –John Zhang, CEO –Dan McDermid • NSERC • UBC – Prof. Andrew Riseman – Prof. David Kitts • North Island College.
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