International Conference on Aquaculture and Fisheries July 20 th -22 nd , 2015 Brisbane,Australia

1. Expression profiles of two downstream signalling molecules of Toll-pathway in Tiger shrimp (Penaeus monodon), Cactus and Dorsal genes in response to infection with white spot syndrome virus and Vibrioharveyi DeepikaAnand, K. Sreedharan,P.P.SureshBabu , AnutoshParia, M. Makesh and K. V. Rajendran ICAR-Central Institute of Fisheries Education (CIFE), Off-Yari Road, Versova, Mumbai-400 061 INDIA International Conference on Aquaculture and Fisheries July 20th-22nd , 2015 Brisbane,Australia

2. Introduction • Diseases affecting shrimps have caused severe losses to the shrimp aquaculture industry • White spot syndrome virus (WSSV)-a devastating pathogen and vibriosis -high mortality in many species of penaeid shrimp • Identification of immune genes and the knowledge of the expression patterns of these genes in the presence and absence of pathogens - role of the immune genes in the shrimp immune system. • To generate protection strategies for sustainable shrimp culture Shrimp immune system Innate immunity - first-line of host defense against pathogens. Activated upon recognition of pathogen associated molecular pattern (PAMPs) such as lipopolysaccharide (LPS), β-1,3-glucans (BG), peptidoglycan (PG) by pathogen recognition recognition ( PRRs) Triggers diverse humoral and cellular activities via signal transduction pathways Major immune signalling pathways - Toll pathway - defense against fungi, Gram-positive bacteria, and viruses  Immune deficiency (IMD) pathway - controlling Gram- negative bacterial infections and virus infection  JAK/STAT pathway - antiviral defense (Li and Xiang 2013)

3. Introduction Components of Toll pathway in shrimp • Spatzle • Toll-like receptor (TLR) (Arts et al., 2007) • Myeloid differentiation factor-88 (MyD88) (Deepika et al., 2014) • Tube (Interleukin-1 receptor-associated kinase-4 homolog) (Watthanasurorotet al., 2012) • Pelle (IRAK-1 homolog) • Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (Deepika et al., 2014) • Cactus (IkB homolog) • Dorsal (NF-kappa B homolog) Li and Xiang 2013

4. Introduction • Rel/NFkappaB -gene transcription in inflammation, immune response, apoptosis, embryonic morphogenesis, cell proliferation and differentiation. • Dorsal belongs to the class II NF-kB family- characteristic RHD domain • Present in the cytosol, forming complex with Cactus, a Drosophila homologue of mammalian IkB proteins. Cactus masks the nuclear localization signal of Dorsal prevents migration into nucleus. • After activation by the Toll pathway in response to infection of fungi or Gram-positive bacteria, the Cactus was degraded and Dorsal translocated into the nucleus to regulate the transcription of such antimicrobial peptide genes Cactus (IkB homolog)and Dorsal (NF-kappa B homolog) from Invertebrates Against this background……

5. The present study… • Partial sequence of Cactus and Dorsal from Penaeus monodon. • Ontogenic expression of PmCactus and PmDorsal in different developmental stages and different tissues ofhealthy P. monodon • Response of PmCactus and PmDorsal against different routes of WSSV and Vibrio harveyi challenges in vivo • Response of PmCactus and PmDorsal upon stimulation with WSSV and bacterial ligands in vitro in primary haemocyte cultures of P. monodon

6. Materials and methods Experimental Animals • Developmental stages-Egg, Nauplii 1-3, Zoea 1-3, Mysis 1-3 and PL1, 2, 3, 4, 5, 7 and 14 • Post larvae –PL-18 • Juvenile – 3 g • Source: Vasavi Hatchery, Kakinada • Transported in RNA later Jiravanichpaisal et al. 2007 • Adult • Size - 25-33 g • Source: Shakthi Aqua Farm Pvt. Ltd. Mumbai • Acclimatised in 15 ‰ sea water and fed daily with a commercial diet at 5% of their body weight. • Tissues collected from 3 animals for normal expression

7. Materials and methods RNA extraction (Trizol reagent, Invitrogen, USA) Quantification (NanoDrop 2000 spectrophotometer, Thermo Scientific, USA) DNase treatment cDNA preparation (RevertAid™ First Strand cDNA Synthesis kit, Thermo Scientific, USA) PCR amplification and cloning  Plasmid extraction (GeneJET plasmid miniprep kit, Thermo Scientific, USA) Sequencing (Eurofins Genomics, Bangalore) 5’RACE (Clonetech Kit, Takara, Japan) Sequencing cDNA cloning • Bioinformatics Tools used for sequence analysis • SMART (simple modular architecture research tool) -to predict the protein domain • BLAST • ORF Finder – to find out the open reading frame within the sequence • Clustal X.2.0 program - Multiple sequence alignments • ExPASy - Translation and protein analysis • Boxshader– shading of the sequence

8. WSSV /Vibrioharveyiinjection Challenge Materials and methods Preparation of primary haemocyte culture and exposure to WSSV • 250 post-larvae (PL18 stage) exposed to • 1 mL of WSSV inoculum • (5.7 x105 copies/mg tissue )/ • 106cfu/mLV. harveyi in • 1L sterile sea water (15 ‰) • 30 animals (average weight- 3g) • exposed to 2.5 mL WSSV inoculum (5.7 x105 copies/mg tissue )/ • 106 cfu/mL • V. harveyiin 3L 15 ‰ seawater • After 3 h of exposure, transferred • to fresh seawater having the • same salinity • 3 pooled PLs / different tissues • of juveniles sampled at 2, 6, 12, • 24, 48 and 72 h post-challenge • Three replicates were used • for each time-point • Uninfected animals were • used as control Immersion challenge with WSSV/ V. harveyi Injection challenge with WSSV/ V. harveyi Surface sterilisation with 70% alcohol Collection of Haemolymph aseptically Centrifugation (400g for 3 min) Resuspension of haemocytes in 2X L-15 medium (with 20% FBS containing antibiotics) and seeding WSSV inoculum (103 copies/µL)/ LPS(10µg) Washing and supplementing with fresh growth media Harvesting at 2, 6 and 12h post exposure WSSV inoculum preparation (Rajendran et al., 1999) Gill and pleopod Tissue samples of WSSV-infected P. monodon, Homogenization in chilled PBS Clarification Filtration Testing virulence In vitro – cold 2X L-15 cell culture medium (Jose et al., 2010) WSSV inoculum -Copy number: 1.3 x 103 copies/µL; V. harveyi- 106 cfu/mL Haemolymph withdrawn from ventral sinus Collection Haemocytes by centrifugation (400g for 3 min) Three replicates used for each time point Tissues collected at 2, 6, 12, 24, 48, and 72 h time points post-injection vv vv vv RNA isolation and cDNA synthesis

9. Materials and methods Real-time quantitative PCR to estimate mRNA expression Template: 100 ngcDNA SYBr Green chemistry ABI 7500 real-time PCR machine The relative fold change determined by the 2-ΔΔCT method (Livak and Schmittgen, 2001) One-way analysis of variance (ANOVA) done using SPSS 16.0 The results expressed as relative mRNA expression levels of mean ± SEM (standard error of the mean) P<0.05 was considered as statistically significant.

10. Results and discussion

11. Results PmCactus 5’ UTR PmCactus cDNA partial sequence – 1534bp (463aa) Ankyrin repeats (ANK domain) PmCactus protein domain topology

12. Results PmCactus Lv-Litopenaeus vannamei (AFO38331); Fc- Fenneropenaeus chinensis; Pm-Penaeus monodon;Ad-Apis dorsata (ACT66871); Mr-Macrobrachium rosenbergii (AET34918); Ll-Lutzomyia longipalpis (ABR28348); Dm-Drosophila melanogaster (AAA85908); Cf- Camponotus floridanus (EFN66754); Dp-Daphnia pulex (EFX89207); Pf-Pinctada fucata (ACF93446); Sk-Saccoglossus kowalevskii (NP_001161607); Mem-Meretrix meretrix (ADK74377); Bm-Bombyx mori (NP_001166191); Hd-Haliotis discus discus (AFO64973); Cg-Crassostrea gigas (ABB52821); Rn-Rattus norvegicus (NP_001099190); Mum-Mus musculus (EDL36726); Bt-Bos Taurus (NP_001039333); Hs-Homo sapiens (NP_065390); Mam-Macaca mulatta (NP_001244679); La-Loxodonta africana (XP_003408908); Cb-Cerapachys biroi (EZA60965) F. chinensis: 97% L. vannamei: 99%

13. Results PmDorsal 5’ UTR PmDorsal cDNA partial sequence – 1155bp (339aa) Rel Homology Domain (RHD) 82-252aa PmDorsal protein domain topology Ig-like, plexins and transcription factors (IPT) domain 257-339aa

14. Results PmDorsal Lv- Litopenaeus vannamei (ACZ98167); Fc-Fenneropenaeus chinensis (ACJ36225); Pm-Penaeus monodon; Ec-Eriocheir sinensis (AHG95994);Tc-Tribolium castaneum (NP_001034507); Dm-Drosophila melanogaster (AAA28479);Ae-Aedes aegypti (XP_001652840);Cq- Culex quinquefasciatus (XP_001844078);Cb- Cerapachys biroi (EZA51108); Nv-Nasonia vitripennis (XP_001602675);Hs- Harpegnathos saltator (XP_011149046); Cf- Camponotus floridanus (EFN68841)   F. chinensis: 99% L. vannamei: 99%

15. Discussion

16. Results and Discussion Gene expression during developmental stages • Jiravanichpaisal et al. 2007 studied the expression of some immune-related genes such as prophenoloxidase (proPO), peroxinectin (Prx), crustin (Crus), penaeidin (Pen), transglutaminase (TGase), haemocyanin (Hc) and astakine (Ak) during larval development of Penaeus monodon, i.e. nauplius 4 (N4), protozoea 1 and 3 (Z1 and 3), mysis 3 (My 3), post-larvae 3 (PL3) and also in haemocytes of juveniles. • Reported that Prx, Crus, Pen, TGase, Hc and Ak were significantly expressed at all larval stages

17. Results and Discussion Tissue-level expression

18. Results Gene expression in post larvae (PL-18)-Immersion challenge with WSSV and Vibrioharveyi

19. Gene expression in gill-Immersion challenge with WSSV and Vibrioharveyi Results

20. Gene expression - WSSV injection challenge Vibrioharveyi Results

21. Gene expression -Vibrioharveyiinjection challenge

22. Results Expression in response to WSSV and LPS in Haemocytesin vitro Primary haemocyte culture WSSV LPS

23. Discussion • Up-regulation of Cactus could be noticed in the scallop, A. irradians in response to Listonella anguillarum except at 3rd and 32 hpi (Mu et al., 2010). • Signifcant up-regulation ofLvCactus could be noticed in the haemocytes of L. vannamei in reponse to V. parahemolyticus challenge, except at 12 h and 48 hpi (Li etal., 2012). • Progressive reduction in the expression level ofLvCactus could be noticed after WSSV challenge (Li et al., 2012). • Wang et al. (2013) noticed that in response to V. alginolyticus, expression level ofFcCactus was significantly up-regulated at 1 and 12 hpi • Kasthuri et al. (2013) noticed up-regulated expression of IkB in the haemocytes of abalone, H. discus discus, in response to V. parahaemolyticus and Listeria monocytogenes, with the highest expression at 72 hpi

24. Discussion • Lu et al. (2013) noticed down-regulation of IkB gene in the sea cucumber, A. japonicus, in response to V. splendidus with the lowest expression at 48 hpi, after that expression level steadily increased and was restored to the normal level. • Down-regulation of FcDorsal could be noticed in haemocytes and lymphoid organ in response to WSSV challenge (except at 1 h and 14 hpi in the case of haemocytes and 2 h and 23 hpi in the case of lymphoid organ) • Significant up-regulation of FcDorsal could be noticed in the haemocytes at 1, 14 and 23 hpi in response to V. anguillarum infection; in lymphoid organ, significant up-regulation could be noticed at 1 and 5 hpi.

25. Conclusion • Ubiquitous ontogenic expression of PmCactus and PmDorsal shows the possible role of these genes during early larval and post larval stages • Sigificantly upregulated expression of these genes post bacterial shows it is more responsive to bacterial infection. • Shrimp infected with WSSV/Vibrios cause significant mortality resulting in major losses. • By studying the immune system and the modulation of immune genes, it is possible for researchers to find enough clues in developing management strategies to control and combat the disease problem in shrimp aquaculture. • In our previous study- provided the experimental evidences to show that Toll-pathway in tiger shrimp, Penaeus monodon responds to white spot syndrome virus (WSSV) through molecular characterization and expression analysis of Toll-like receptor (TLR) gene and two essential molecules involved in the Toll-pathway, myeloid differentiation factor 88 (MyD88) and tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) (Deepika et al. 2014) • The data will provide more evidence for understanding the role of Rel/NF-kappaB in the immune system of shrimp.

26. Acknowledgements National Fund for Basic, Strategic and Frontier Application Research in Agriculture (NFBSFARA), ICAR Director, Central Institute of Fisheries Education (CIFE), Mumbai

27. Thank You

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