Palinurid Lobster Larvae. Presented by: Mike Hall

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1 Diet & Microbial Interactions ti In Palinurid Lobster Larvae Presented by: Mike Hall

2 Marine Lobsters Nephropoidea Palinuridea Scyllaridae Various Jasus spp. Panulirus cygnus Tropical Panulirus spp. Homarus americanus Panulirus argus Panulirus argus Nehrops norvegicus

3 Lobster Biodiversity Species: Wild Fisheries and Aquaculture Potential Group Species Wild Fishery Major Minor/ None Aquaculture Potential Seafood Aquarium Family Palinuroidea Family Scyllaridae Subfamily Arctidinae 16 - Some ++ + Subfamily Ibacinae Subfamily Scyllarinae Subfamily Theninae Some Family Synaxidae Family Nephropoidea Subfamily Neophoberinae Subfamily Thymopinae Subfamily Nephropinae 26 3 Some +(++) + Family Thaumastochelidae Family Enoplometopoidae TOTAL Some Several Some

4 Seafood Groups as Production Units Price per kg per whole animal Fish Crustacean Molluscs Cephalopods US $/kg European Lobster American Lobster Mantis shrimp Whiteleg shrimp Rose shrimp Black Tiger Shrimp c/w Octopus Sepia Squid Carp Nile Perch Trout Salmon Eel Misc Mackerel Sprat Swordfish Skipjack Yellowfin Bluefin Sole Turbot Cod 0 Clam Scallop Oyster Mussel Spiny Lo lob obster Source: Globefish, European Price Report 2008

5 Australian Rock Lobster Biodiversity Potential Aquaculture Candidates (9 species) P. polyphagus P. homarus P. versicolor P. ornatus $22 mill. P. penicillatus P. longipes P. cygnus $281 mill. J. edwardsii $152 mill. J. verreauxi $5.4 mill.

6 Model of Aquaculture Propagation System Closed life cycle Wild stock Broodstock Maturation ti Harvesting Processing Marketing Closed-life Cycle (Farming) Closed-cycle l Spawning Farming Independent of wild stock Selective breeding Biosecurity Domestication Grow- out Hatching Larval Rearing

7 Closed-life cycle Propagation System Wild stock (Captured Rearing) Broodstock Maturation Spawning Harvesting Processing Marketing Growout? Larval Rearing

8 Larval Periods of Selected Crustacea Panulirus ornatus P. cygnus Jasus verreauxi J. edwardsii Homarus Crabs Cherax spp. Penaeus spp. Aquaculture Species Time (months)

9 Palinuridae Phyllosoma Larval Rearing Larval Cycle Completed in > 9 species Species Panulirus species Reference Panulirus argus Moe (1991), Goldstein et al. (2008) Panulirus elephas Mercer et al. (1997), Kittaka et al. (1988), Kittaka et al. (2000) Panulirus homarus Radhakrishnan and Vijayakumaran (1993) Panulirus japonicus Oshima (1936), Kittaka and Kimura (1989), Yamakawa et al. (1989), Sekine et al. (2000) Panulirus interruptus Johnson (1956), Dexter (1972) Panulirus lalandii Kittaka (1988) Panulirus longipes Matsuda and Yamakawa (2000) Panulirus ornatus MGKailis (2006), AIMS (2007) Panulirus pencillatus Matsuda et al. (2006) Panulirus polyphagus Saisho (1966), Sin (1967) Jasus edwardsii Jasus species Kittaka et al. (1988), Booth (1996), Illingworth et al. (1997), Tong (1997), Moss (2000), TAFI (2005) Jasus verreauxi Kiattaka et al. (1997), TAFI (2006) Thenus orientalis Mikami and Greenwood (1997) Scyllarid species

10 Smith, G., Salmon, M., Kenway, M. and Hall, M. (2009) Description of the larval morphology of captive reared Panulirus ornatus spiny lobsters, benchmarked against wild caught specimens. Aquaculture 295:76-88.

11 Change in Larval Size through Development 11 Stages

12 Model of Larval Survival Continuous larval attrition and mass mortality events 100,000 90,000 80,000 Mortality/month 10% 6 month larval phase Progen ny 70,000 60, ,000 40,000 30,000 20,000 10, % 30% 40% 50% Months 10% Survival

13 Conceptual Model Interactions between Nutrition Health - Growth 6 Metabolomics Healthomics Nutrition Health Growth Nutrigenomics Microbiomics

14 Nutrition Disease - Growth 6 Nutrition Health Growth Growth

15 Poor Nutrition Health - Growth 6 Nutrition Health Growth Growth

16 Poor Nutrition Disease - Growth 6 Nutrition Health Growth Morbidity Death Growth

17 Optimising the Triad Interactions between Nutrition Disease - Growth 6 Metabolomics Healthomics Nutrition Health Growth Nutrigenomics Microbiomics

18 Microbial Inputs Microbial Biosecurity Primary Input 1 Seawater 2 Added daily 2. Feed Artemia 60:1 x 100 s days Feed Hatchery Tank 3 Biofilm Planktonic bacteria 4 Phyllosomas Continuous Added once 3. Biofilm Complex community 4. Phyllosomas 90% of cells are bacteria Random Other vectors Aerosols, staff Discharge

19 Microbial Sources and Sinks in Hatchery Four Compartments 1. Seawater Column 2. Larval Feeds 3. Biofilm 4. Phyllosoma External Internal

20 Biofilm Complex Bacterial Ecosystem Diving through the biofilm Surface to Tank (2 µm steps)

21 Interactions between Nutrition Disease - Growth Larval finfish consume feed whole - Feed size related to mouth gape Source: Artemia World

22 Interactions between Nutrition Disease - Growth Phyllosomas consume feed piecemeal Stage 1 Stage 7

23 Health - External Fouling Mandibles Paragnaths 1st Maxilla Endites Basal Coxal

24 Changes in Mouthpart through Development Stages 1 to 11 Stage 1 Stage 11

25 Larval phyllosomas consume feed piecemeal Feeding video artemia

26 External Bacterial Fouling of Phyllosoma Mouthparts Day 1 Day 3 Post-molt Post-molt

27 External Bacterial Fouling of Phyllosoma Mouthparts Day 11 Post-molt Day 13 Post-molt FISH probe - Thiothrix Immediate Post-molt

28 Fouling (Thiothrix) of Phyllosoma Mouthparts Stage 1 to 2 ree of Fo ouling Deg S1 to S2 Typical Moult Cycle 7days Day since Moult

29 Fouling (Thiothrix) of Phyllosoma Mouthparts Stage 3 to 4 compared to Stage 1 to 2 Deg gree of Fouling S1 to S2 S3 to S4 Typical Moult Cycle 7 days Day since Moult

30 Interactions between Nutrition Health - Growth Fouled Hatchery compared to Unfouled FOULED WILD Body Length (m mm) Developmental Stage

31 Interactions between Nutrition Health - Growth Unfouled Hatchery compared to Wild 35.0 WILD HATCHERY Body Length (m mm) Developmental l Stage

32 Interactions between Nutrition Health Growth Internal Infection

33 Interactions between Nutrition Health Growth Ingestion and Digestion

34 Interactions between Nutrition Health Growth Internal Infection Wild-caught larva with intact hepatopancreas Hatchery larva with white-gut syndrome, infected by Vibrio spp.

35 Phyllosoma gut infection Real-time video

36 Health Internal Infections Survival of P. ornatus phyllosomas (S5) challenged with Vibrio sp. (DY05) Surv vival (%) Time (Days) Control DY05

37 Phyllosoma gut Internal bacterial colonization Healthy Moribund Histopathology of hepatopancreas (H&E) Healthy: caeca open and clear Moribound: caeca are blocked and disrupted

38 Fluorescent in situ hybridisation (FISH) Bacterial localisation Healthy Moribund Probes: Red = general bacteria, Purple = Vibrio

39 Bacterial Diversity in Hatchery Phyllosoma Clone Library Analysis Only single representatives: Rare Operational Taxonomic Units (OTUs) Thiothrix Vibrio Bacteroidetes Photobacterium Sulfitobacter Thiothrix Vibrio Bacteroidetes Photobacterium Sulfitobacter Episilonbacter Roseobacter Exiguobacterium Psychrobacter Single OTUs

40 Interactions between Nutrition Health - Growth Oceanic Hunting Probiotics Tank Health Natural Microbial community Health Probiotic Microbial community

41 Larval Ecology of P.ornatus in NE Australia Phyllosoma entrained in the Coral Sea Gyre

42 Bacterial Diversity in Wild Phyllosoma Clone Library Analysis Psychrobacter Only single representatives: Rare Operational Taxonomic Units (OTUs) Sulfitobacter Episilonbacter il Thiothrix Vibrio Bacteroidetes Photobacterium Sulfitobacter Episilonbacter Roseobacter Exiguobacterium Psychrobacter Single OTUs Exiguobacterium Roseobacter

43 Bacterial Community Composition Hatchery vs Wild Hatchery Wild External Internal Internal Thiothrix Vibrio Bacteroidetes Photobacterium Sulfitobacter Epsilonbacteria i Roseobacter Exiguobacterium Psychrobacter Single OTUs % Bacterial Clone Library Representation

44 The search for Probiotics Well diffusion assay agar infused with strain DY05 Homogenise Replica plating Sample natural environment 16S rdna sequencing Identification + AIMS culture collection Well Diffusion Assay Target organism: Vibrio sp. DY05

45 Sourcing Probiotic Strains 18% (92/508) of isolates inhibited growth of known P. ornatus pathogens (Vibrio strains DY05 & D40) in well-diffusion assays 4% 2% 1% 2% Zooplankton 39% 52% Wild Phyllosomas Hatchery Phyllosomas AIMS Hatchery Seawater Other

46 Identifying the Probiotic Candidates 16S rdna sequencing of 60 candidate probionts Psychrobater Roseobacter 13% Bacillus 3% 2% 2% 2%2% Vibrio Pseudoaltermonas 76% Pseudoaltermonas Vibrio Roseobacter Psychrobacter Bacillus Ferrimonas Acinetobacter

47 Current and future direction Interactions ti between Nutrition Health - Growth Elucidation of pathogen-probiont p interactions in vivo Vibrio (DY05) Infection GFP labelled Vibrio (DY05) At Artemia gut

48 Closed life cycle Aquaculture Panulirus us ornatus Wild stock Broodstock Maturation ti Spawning Hatching Harvesting Processing Marketing Grow- out Larval Rearing

49 Wild stock (Captured Rearing) Closed life cycle Aquaculture Panulirus ornatus Broodstock Maturation Closed-life Cycle (Farming) Spawning Harvesting Processing Marketing Growout Larval Rearing

50 Staff Investment Area Water Quality Broodstock Facility Larval Rearing Facility Live Feed Facility Health (Microbiology) Research Diet (Larval Feeds) Research Cruises Natural History, Prey, Probiotics, Diet Associated Larval Research Staff Matt Kenway Jane Gioffre Michael Clarkson Katie Holroyd Matt Salmon Grant Milton Justin Hochen Greg Smith Lone Høj Mike Hall Greg Smith Mike Hall Above staff plus volunteers 6 PhDs, 1 MSc Evan Goulden Ana Cano Gomez