JAK/STAT SIGNALING PATHWAY IN ARTEMIA FRANCISCANA EXPRESSION OF SELECTED JAK/STAT SIGNALING PATHWAY AND IMMUNE-RELATED GENES UPON POLY(I:C) PRETREATMENT AND VIBRIO PATHOGEN CHALLENGE OF GNOTOBIOTIC BRINE SHRIMP Ryan John P. Pascual Student number: 01700920 Promotor: Prof. Dr. Ir. Peter Bossier Tutors: Mr. Biao Han Dr. Ir. Stephanie de Vos Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Aquaculture Academiejaar: 2018 - 2019 The author and the promoter give the permission to use this master dissertation for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws, more specifically the source must be extensively when using from this thesis. De auteur en de promotor geven de toelating deze masterproef voor consultatie beschikbaar te stellen en delen van de masterproef te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting de bron uitdrukkelijk te vermelden bij het aanhalen van resultaten uit deze masterproef. Gent, 23 August 2019 The promoter, The author, Prof. Dr. Ir. Peter Bossier Ryan John P. Pascual i TABLE OF CONTENTS Copyright i Table of Contents ii List of Tables iv List of Figures v List of Appendices vii List of Appendix Tables viii List of Appendix Figures ix List of Acronyms x Abstract xi Chapter 1. Introduction 1 Chapter 2. Review of Related Literature 3 2.1. Artemia as a Crustacean Model 3 2.2. JAK/STAT Pathway and its role in Drosophila Immunity 8 2.3. Crustacean JAK/STAT Pathway Genes 12 2.4. Polyinosinic:polycytidilic acid and nucleic acid-induced immunity in shrimp 16 2.5. Vibrio harveyi 18 Chapter 3. Methodology 21 3.1. In silico determination and characterization of Artemia franciscana JAK/STAT 21 signaling pathway genes 3.2. Bacterial strains and culture conditions 25 3.3. Gnotobiotic Artemia nauplii culture 26 3.4. Toxicity of poly(I:C) in gnotobiotic Artemia 26 3.5. Effect of poly(I:C) exposure on survival of brine shrimp upon pathogen challenge 26 3.6. Effect of poly(I:C) exposure and Vibrio harveyi pathogen challenge on the 27 expression of selected JAK/STAT pathway and immune genes 3.7. Statistical Analyses 32 Chapter 4. Results 33 4.1. In silico determination and characterization of Artemia JAK-STAT 33 signaling pathway genes 4.2. Toxicity of poly(I:C) in gnotobiotic Artemia 56 4.3. Effects of poly(I:C) exposure on survival of brine shrimp upon pathogen challenge 56 4.4. Expression of selected JAK/STAT signaling pathway and immune-related genes 60 Chapter 5. Discussion 67 5.1. JAK-STAT Signaling Pathway in Artemia 67 5.2. Toxicity and Protective Effect of Poly(I:C) Exposure and Pretreatment 70 5.3. Expression of selected JAK/STAT signaling pathway and immune-related genes 72 ii Chapter 6. Conclusion 78 Chapter 7. Future Perspectives 80 Literature Cited 83 Appendices 95 Acknowledgements 123 Biographical Sketch 124 iii LIST OF TABLES 1 Copy number of JAK-STAT pathway signaling genes among representative 12 arthropod species 2 Sequenced JAK-STAT signaling pathway genes reported in crustaceans 13 3 List of Annotated Arthropod JAK-STAT pathway genes used in the search of 22 JAK-STAT genes in the Artemia genome and transcriptome 4 List of PROMO transcription factors used in this study 25 5 Sequences of primers used in this study 30 6 Artemia franciscana JAK/STAT gene models with significant BLAST results 34 7 Reciprocal BLAST in NCBI database of identified Artemia franciscana JAK/STAT 39 gene models 8 Amino acid sequence similarity and identity of putative AfSOCS6 (partial) with 48 reported SOCS6 and SOCS7 sequences 9 Amino acid sequence similarity and identity of putative AfVago with reported 51 L. vannamei Vago proteins 10 Predicted transcription factor binding sites of Artemia STAT-activated genes 54 iv LIST OF FIGURES 1 Arthropod phylogeny with focus on Pancrustacea 4 2 Life cycle of sexually reproducing Artemia 6 3 Research applications of gnotobiotic animals 7 4 General mechanism of the JAK-STAT Pathway 9 5 Schematic diagram of the Drosophila JAK/STAT pathway 10 6 Schematic diagram of recently described mechanisms of crustacean JAK-STAT 15 signaling 7 Schematic structure of Polyinosinic-polycytidilic acid or Poly (I:C) 16 8 Vibrio harveyi morphology and gross signs of infection in laboratory infected 18 Litopenaeus vannamei shrimp 9 Experimental design of gene expression study 29 10 Phylogenetic tree analysis of JAK proteins from various species 42 11 Phylogenetic tree analysis of STAT proteins from various species 43 12 Phylogenetic tree analysis and domain structure analysis of DOME proteins 45 from various species 13 Phylogenetic tree analysis of SOCS proteins from various species 46 14 Domain structure analysis of the putative AfSOCS proteins 47 15 Multiple sequence alignment of SOCS6 proteins from various species 49 16 Multiple sequence alignment of Vago proteins from various species 50 17 Phylogenetic tree analysis and domain structure analysis of Vago proteins from 51 various species 18 Phylogenetic tree analysis and domain structure analysis of Dicer proteins 52 from various species 19 Sequence logos of the predicted binding site of PROMO STAT transcription 55 factors in the untranslated upstream sequence of selected JAK-STAT activated genes v 20 Percent survival of axenic Artemia nauplii after 48 hours exposure in varying 56 concentration of Poly(I:C) 21 Effect of Poly(I:C) pretreatment (2h exposure: 2h recovery) on the survival of 57 unchallenged and Vibrio harveyi BB120-challenged gnotobiotic brine shrimp 22 Effect of Poly(I:C) pretreatment (2h exposure: 2h recovery) on gnotobiotic 58 artemia survival after 48 hours of Vibrio harveyi BB120 challenge 23 Effect of pretreatment and bacterial challenge on survival in FASW and Shrimp 59 RAS challenge media 24 Effect of pretreatment and bacterial challenge on survival of axenic and non- 59 axenic brine shrimp nauplii 25 Validation of amplicon size of primers used in this study (1.75% agar, 0.5x TBE) 60 26 Relative gene expression of pretreated and untreated brine shrimp at 0h 61 27 Relative gene expression of STAT 62 28 Relative gene expression of SOCS6 and Dome 63 29 Relative gene expression of Vago and Dicer-2 64 30 Relative gene expression of Prophenoloxidase (ProPO) and Transglutaminase2 66 (Tgase2) 31 Comparison of the generalized JAK/STAT signaling pathway in Drosophila, 68 Artemia, and penaeid shrimp vi LIST OF APPENDICES 1 Toxicity of Poly(I:C) in gnotobiotic brine shrimp 95 2 Survival of gnotobiotic brine shrimp exposed to varying concentrations 96 of Poly(I:C) 3 Survival of gnotobiotic brine shrimp with Poly(I:C) pretreatment during 97 Vibrio harveyi BB120 challenge in FASW and Shrimp RAS as challenge media 4 Survival of axenic and non-axenic brine shrimp with Poly(I:C) pretreatment 98 during Vibrio harveyi BB120 challenge 5 Sequences of selected gene models and analysis of transcription factor binding 99 sites 6 Validation of designed Quantitative PCR primers 113 7 Determination of the optimal dilution of cDNA for qPCR analysis 122 vii LIST OF APPENDIX TABLES 1 Predicted transcription binding sites of known STAT-activated developmental 102 genes (USH, Eve and Raf) present in Artemia franciscana 2 Predicted transcription binding sites of Artemia fransciscana STAT-activated 104 genes 3 Melting temperature and melt curve of QPCR products of the primers 120 designed for this study viii LIST OF APPENDIX FIGURES 1 Repeated experiment on the percent survival of axenic Artemia nauplii after 48 95 hours exposure in varying concentration of Poly(I:C) 2 Repeated experiment on the effect of Poly(I:C) pretreatment (2h exposure: 2h 96 recovery) on gnotobiotic artemia survival after 48 hours of Vibrio harveyi BB120 challenge 3 Repeated experiment on the effect of Poly(I:C) pretreatment (2h exposure: 2h 96 recovery) on gnotobiotic artemia survival after 48 hours of Vibrio harveyi BB120 challenge 4 Repeated experiment on the effect of pretreatment and bacterial challenge on 97 survival in FASW and Shrimp RAS challenge media 5 Repeated experiment on the effect of pretreatment and bacterial challenge on 98 survival of axenic and non-axenic brine shrimp nauplii 6 Amplification and melt curve of cT determination experiment using four 122 dilutions of the same sample and four primers (GAPDH, EF-1, ProPO and Tgase2) ix LIST OF ACRONYMS BLAST Basic Local Alignment Search Tool cT Cycle threshold DOME Domeless dsRNA Double stranded RNA EF-1 Elongation Factor 1 Et Eye transform E-value Expect value FASW Filtered autoclaved artificial seawater GAPDH Glyeraldehyde-3-Phosphate-Dehydrogenase Hop Hopscotch JAK Janus kinase JAK/STAT Janus kinase-Signal transduction and activator of transcription PCR Polymerase Chain Reaction PIAS Protein inhibitor if activated STAT Poly(I:C) Polyinosinic-polycytidilic acid ProPO Prophenoloxidase PTP Phosphotyprosine phosphatase qPCR Quantitative Polymerase Chain Reaction RAS Recirculated aquaculture system SOCS Suppressor of Cytokine Signaling STAT Signal transduction and activator of transcription Tgase2 Transglutaminase 2 Upd Unpaired WSSV White spot syndrome virus x ABSTRACT PASCUAL, RYAN JOHN PALUSTRE. Universiteit Gent. August 2019. JAK/STAT Signaling Pathway in Artemia franciscana. Expression of selected JAK/STAT signaling pathway genes upon Poly(I:C) pretreatment and Vibrio pathogen challenge of gnotobiotic brine shrimp. The Janus kinase-Signal transduction and activator of transcription (JAK/STAT) signaling pathway is important in antiviral immunity, as well as other biological processes. Analysis of available genomic data on brine shrimp (Artemia franciscana) was used to determine the components of the brine shrimp JAK/STAT signaling pathway. Majority of the core components and pathway regulators found in fruit fly and penaeid shrimp were identified to be present in brine shrimp.