Sea Lice Research Centre SLRC update
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1 Sea Lice Research Centre SLRC update Christiane Eichner University of Bergen
2 SeaLiceResearchCenter (SLRC) Centre for Research-based Innovation
3 What is an SFI? SFI = Centre for Research based Innovation One of three centre funding from Norwegian Research Council (NRC) Purpose: build up and strengthen Norwegian research groups that work in close collaboration with partners from innovative industry and innovative public enterprises Selection based on free competition between diverse applications NRC provides 5+3 years of funding (~1.3 mill Euros/year), requires 50% own funding (> 25% industrial funding)
4 9 Partners in SLRC Academic Partners: University of Bergen Norwegian School of Veterinary Science Institute of Marine Research UNI Research AS Industrial Partners: Novartis Animal Health AG EWOS Innovation AS Patogen Analyse AS Marine Harvest ASA Lerøy Seafood Group ASA
5 Goal for SLRC Research to develop new control measures against sea lice. Main goal: The Sea Lice Research Centre aims at becoming world leading on research on salmon louse and related parasites. The nature of the centre will facilitate development of new methods for lice control and shorten the time from basic research to new products and tools for parasite control in the aquaculture sector to achieve a true integrated pest management in the future.
6 Organisation Board of Directors representative of each Partnerorganisation SAB Scientific Advisory Board Centre Leader Project manager Leader Group WP-leaders & Centre Leader WP1 WP2 WP3 WP4 WP5 WP6
7 SLRC Leadership Centre Director WP1 Medicine & Resistancy WP2 Antiattachment University of Bergen Department of Biology WP3 Immunomodulation Prof. Frank Nilsen WP4 Molecular parasitology (new control methods) Prof. Tor E. Horsberg WP5 LiceBase (genome recources/ integrated database) Dr. Simon Wadsworth Prof. Øystein Evensen WP 6 LiceLab (wetlabs, experimental facility) Prof. Rune Male Prof. Inge Jonassen Dr. Sussie Dalvin Lars Hamre
8 Interaction of the different WPs WP1 Chemotherapeutants & resistance WP2 Anti attachment WP3 Immunomodulation WP4 Molecular parasitology WP5 LiceBase WP6 LiceLab Salmon louse Genome sequence
9 Improved Sea Lice control Salmon louse biology New Medicines Feed supplements Salmon louse genome Innovation for Improved Lice control Immune control Wet-lab facility
10 Salmon louse genome (WP5) Inbred strain High Throughput sequencing assembly ESTs + RNAseq annotation LiceBase: annotation & establish an integrative database resource
11 Benefits for SLRC-researcher Interesting features in salmon louse genome that help to understand its biology Easy to use tools which save an enormous amount of time-consuming work for molecular biologists e.g. interesting gene known from other organism blast against genome yes/no find genomic sequence, predicted EST, protein sequence, splice variants, orthologs, paralogs,.. Or: finding genes not known from other organisms
12 WP5: Bioinformatics Determine full length sequence Copy number of candidate gene Analyze homologs in related organisms Determine gene repertoire for different molecule synthesis systems Map metabolic pathways Identify stage/organ specific transcripts (from RNA sequencing of selected stages and isolated organs)
13 Gene-tool: EnsemblMetazoa Ensembl Genomes is developed by the European Bioinformatics Institute (Part of the European Molecular Biology Laboratory) EMBL-EBI and is powered by Ensembl software system for the analysis and visualisation of genomic data. genomic sequence EST protein sequence splice variants Orthologs/paralogs...
14 Gene-tool: EnsemblMetazoa University of Bergen Department of Biology
15 Gene tree University of Bergen Department of Biology
16 RNA-sequencing Shows in which stages interesting genes are expressed Helps to get more information about unknown genes
17 Wet Lab facilities at SLRC (WP6) 1x1 meter tanks designed to maintain lice strains as material for research general test efficacy of treatments: RNAi, vaccines, drugs...
18 Wet Lab facilities at SLRC (WP6) Single fish tanks Efficacy assays (vaccine, drugs, feed) Selective breeding o monitor loss of lice from individual fish o screening of many RNAi targets using a minimum of fish and space
19 Wet Lab facilities at SLRC (WP6) Hatchery (flow through) University of Bergen Department of Biology Wet table with 100 outlets Room for mm incubators (each labeled with unique identity)
20 Wet Lab facilities at SLRC (WP6) Additional capacity at ILAB and BIO 500 litre tanks University of Bergen Department of Biology
21 Wet Lab facilities at SLRC (WP6) LiceLab at UIB 10 1x1 meter tanks 114 single fish tanks (improved design) Hatchery (flow through); room for mm incubators additional capacity at ILAB and BIO when required (500 litre tanks) LiceLab at IMR 48 single fish tanks Hatchery: incubators can be run simultaneously with variable salinity and temperature (flow through) 160 litre, 250 litre and 500 litre fish tanks: salinity and temperature can be mixed in each individual tank. LiceLab EWOS Dirdal 16 tanks (500 litre) Hatchery (stagnant water)
22 Lice material (WP6) 6 strains of lice maintained resistant strain Why more lice when there are so many lice around? Availability of lice from right stage at right time assured Comparable experimental material Enough experimental material at given time point
23 RNA inteference (WP6) A process in which the introduction of double-stranded RNA into a cell inhibits the expression of genes. (naturally or artificially introduced) Sequence specific (choose gene to be silenced) Silencing lasts from 2 days to at least 2 month Information about the function of a gene Simulation of vaccine or drug: targets
24 RNAi screening: Finding targets (WP6) Goal: identify genes that encode proteins that are essential and unique! Salmon louse genome ~ genes. Screen of 100 genes/year (2012, 2013) Sequence analysis Exclude conserved genes Look for secreted proteins Unknowns (30-40% have little or no information) Expression patterns (RNA-seq) Selection based on when proteins are used
25 Preadult/adult RNAi screen collect preadult lice (timed infection) Injection of dsrna Lice on fish (single fish tanks) Sample adult lice: investigate and incubate eggs Investigate offspring 30 with abnormal phenotype Four candidate genes to be produced for test vaccines (Novartis)
26 Effective RNAi methode for nauplia screen Preadult/adult screen: labour intensive, small part of the life cycle Nauplia screen: whole lifecycle, both sexes
27 Nauplius screen Incubation in double stranded RNA during molt Sucessfull silencing nonviable phenotype Longelivity is in test now
28 Candidate gene screening Lice Genome LiceBase (WP5) RNAi screen Evaluation as drug target Evaluation as feed supplements Phenotype assessment Evaluation as vaccine target LiceLab (WP6) Evaluation as bioactive molecules Test vaccine Candidates for commercial exploration
29 WP4 Molecular parasitology and host parasite interactions: University of Bergen Department of Biology Identification and evaluation of targets for immune control and vaccine development. Attachment Feeding, digestion, nutritional status Host recognition Exocrine regulation Sex differentiation and maturation Endocrine regulation Molting Reproduction Germ cell differentiation, gonad establishment, meiosis and maturation to gamets
30 WP4 Molecular parasitology and host parasite interactions: Identification and evaluation of targets for immune control and vaccine development. Copepodid biology Chemosensory system Gene regulation in parasitic copepodids Reproduction, germ cell differentiation and maturation Nuclear receptors Germline formation Oocyt maturation Endo and exocrine system in salmon louse
31 Finding ligands of receptors: Ligand: Any substance (e.g. hormone, drug, functional group, etc.) that binds specifically and reversibly to another chemical entity to form a larger complex. A ligand may function as agonist or antagonist. Receptors chemosensory system (attraction/repulsion) Endocrine system (hormones; metabolism, growth and development) How to test possible ligands? Xenopus oocyte system (Novartis, Saint Aubin) Two-hybrid Ligand activation assay
32 Sensory system of copepodites (WP4 & WP2) Xenopus oocyte system (Novartis, Saint Aubin) Finding substances that could interrupt the infestation of the copepodites Specific ligand Voltage measurement 27 ligands tested (Different concentration, time)
33 Hormone receptor ligands Hormones: Molting, Reproduction Two-hybrid Ligand activation assay Activation domain NR1-LBD Gal4 DNA Binding Domain NR2/3 LBD & DBD Finding substances that could interrupt important processes in the salmon louse RNAPII 5xGal4_RE luciferase
34 Salmon lice glands Numerous glands Different morphology Modulation of host response? Bell et al DAB-staining - exocrine glands Tadiso, T. M., et al 2011 Bmc Genomics 12. Possible target for vaccines/new drugs
35 Finding genes expressed in this glands Finding candidate genes in the genomic ressources starting from immunmodulating substances known in other organisms Localization in the salmon louse by in situ hybridisation Pre-adult female RNAi with candidate Less infestation???
36 Life cycle of the salmon louse Wet-lab facilities gave possibilities for inspecting the life cycle of the salmon louse in a new way Investigation of development and molting in early infested stages is impossible by sampling wild lice. Flow-through system: survival and molting of lice off the fish possible; observation of individual lice in single incubators
37 Schram, 1993 University of Bergen Department of Biology Adjustment of the life cycle of L. salmonis Copepodit Young Ch1 I Old Ch1 Chalimus II Young Ch2 I Nauplius II Adult II I Pre-adult Old Ch2
38 New life cycle of L. salmonis II Copepodit I Nauplius Chalimus I II Hamre, L. A., Eichner, C., Caipang, C. M. A., Dalvin, S. T., Bron, J. E., Nilsen, F., Boxshall, G., and Skern-Mauritzen, R., The salmon louse lepeophtheirus salmonis (copepoda: caligidae) life cycle has only two chalimus stages. Plos One 8, e doi: /journal.pone Adult II I Pre-adult
39 Conclusions WP5: LiceBase (genome recources/ integrated database) The whole genome is sequenced, assembled and annotated available for SLRC-researcher and soon be published WP6: LiceLab Wet-lab facilities are expanded and improved for better reproducibility and higher turnover Candidate genes to be produced for test vaccines identified by RNA interference screening screening method for nauplia screen established (whole lifecycle, both sexes, higher throughput)
40 Conclusions WP4&2 (Molecular parasitology, Antiattachment) progress in investigation of the endocrine, exocrine and sensory system (foundation for target search). WP1 (Medicine & Resistancy) Prof. Tor. E. Horsberg WP3 (Immunomodulation) (NVH) investigation of immunemodulation on the fish site in cell culture experiments on fish at LiceLab facilities going on as well
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