Deepika Anand, K. Sreedharan,P.P.Suresh Babu , Anutosh Paria, 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 FisheriesJuly 20th-22nd , 2015Brisbane,Australia

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 Vibrio harveyi

IntroductionDiseases 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

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)

Shrimp immune system

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) (Watthanasurorot et 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

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

Introduction

Cactus

Pacific oyster, Crassostrea gigas Montagnani et al., 2008Pearl oyster, Pinctada fucata Xiong et al., 2008Scallop, Argopecten irradians Mu et al., 2010Pacific white shrimp, Litopenaeus vannamei Li et al., 2012Sea cucumber, Apostichopus japonicus Lu et al., 2013Abalone, Haliotis discus discus Kasthuri et al., 2013Chinese shrimp, Fenneropenaeus chinensis Wang et al., 2013

Dorsal F. chinensis Li et al., 2010L. vannamei Huang et al., 2010

Cactus (IkB homolog) and Dorsal (NF-kappa B homolog) from Invertebrates

Against this background……

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 of healthy 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

•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

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

Experimental Animals

Materials and methods

Jiravanichpaisal et al. 2007

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

Materials and methods

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

Primers used in the study Gene cloning Primers

PmCactus F1 TTCGGATCAACTCAGCTC

PmCactus R1 CTCCAATCCTGAGGTCATC

PmDorsal F1 TCTGGAGAGCAAACGTAAAG

PmDorsal R1 GCACGTACACCTTTATGG

5’ RACE Primers

PmCactus-5’-R1 GAGGAGGTCGTATCGTTTTCGGGCGT

PmCactus-5’- NR1 AGGTAGTAGAGGAGGCCAGGTGAAG

PmDorsal-5’-R1 GACACAGGACACCACCACCACAGCA

PmDorsal-5’-NR1 CCTACCACCTGAATAGCTGGGTAAGT

cDNA cloning

Materials and methods

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)

 

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 postexposure

Preparation of primary haemocyte culture and exposure to WSSV

vv

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

Injection challenge with WSSV/ V. harveyi

RNA isolation and cDNA synthesis

WSSV /Vibrio harveyi injection Challenge

vv

•250 post-larvae (PL18 stage) exposed to

1 mL of WSSV inoculum (5.7 x105 copies/mg tissue )/

106cfu/mL V. 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. harveyi in 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

vv

Immersion challenge with WSSV/ V. harveyi

Template: 100 ng cDNA

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.

Real-time quantitative PCR to estimate mRNA expression

Materials and methods

Real-time PCR primers

EF-1α-F GGTGCTGGACAAGCTGAAGGC

EF-1α-R CGTTCCGGTGATCATGTTCTTGATG

PmCactusQF TTCGGATCAACTCAGCTC

PmCactus QR CTCCAATCCTGAGGTCATC

PmDorsal QF GGGTCGAGTGCCAAGTATGC

PmDorsal QR GCGATCTCGAGCACATTCTTC

Results and discussion

5’ UTR

Ankyrin repeats (ANK

domain)

PmCactusResults

PmCactus cDNA partial sequence – 1534bp (463aa)

PmCactus protein domain topology

acacacgctc agtagacttcactgggatcagacgtaggaagtacggcattgagtg gttatgtttatggtggtgcttgtctagatgaagtggttcagtgtt ttggtttatagcttggccatgtgtgtgccttagtgagtttactat

146 ATGCCGATAGAGGTAACTAACGAAATTGTACCCACTGAAGAGTGG M P I E V T N E I V P T E E W 191 TACTGGGGCGTATTGATCTTGTGTGATAGTCTGTTTACACCTGGA Y W G V L I L C D S L F T P G 236 CTCTATGCTAAGATGTGGCACATTGGCAGTGCCCAGGCTCAGGGC L Y A K M W H I G S A Q A Q G 281 GGCGTAGGGGAGACGGCAGTAGGATCGGGGTTTGCCTTCGGCTCC G V G E T A V G S G F A F G S 326 CCTATGTCACAAGGGAGAGACGAGGGTGCCAGGCCCAAGGTTTCA P M S Q G R D E G A R P K V S 371 TCCCCTACTACAGACTGCAAATCATACTACGACTCTCACAACGAC S P T T D C K S Y Y D S H N D 416 TCGGGTTTCCTGTCTGGTTCAAACCTTGTGTCCTCCTCGAGTCTA S G F L S G S N L V S S S S L 461 AGTTGTGAGGATAACTCCACCATGCGGTGTAAGGAATCCACGTCC S C E D N S T M R C K E S T S 506 CCTCAGGAGGGCCACAGAAGCCCTGACCCCAAGACTCAGGGCATC P Q E G H R S P D P K T Q G I 551 ACCTCTGTGGGGCATCTCGATTCCGGCATCGACATTTCGGATCAA T S V G H L D S G I D I S D Q 596 CTCAGCTCTCTTCACCTGGCCTCCTCTACTACCTCATCCTCGAGC L S S L H L A S S T T S S S S 641 ACGACGTCAGATACGCCCGAAAACGATACGACCTCCTCCAAGAAG T T S D T P E N D T T S S K K 686 GTTTCGGCGCCACCACGGCGATCTTGCCTCGAGTTAAACGAAGCT V S A P P R R S C L E L N E A 731 CAGTTAGCGCTCCTTCAAGAGATATTTCAGCGAGATGAAGACGGA Q L A L L Q E I F Q R D E D G 776 GACACTCAGCTGCACGTGGCGGTCATGCGTGGCTTCGTCGAGGTG D T Q L H V A V M R G F V E V 821 GTGTACCACATCACGCGACTCCTCCCCCACCAGGCGCTGCTCGAT V Y H I T R L L P H Q A L L D 866 CTCGCCAACCACACCGGCAGGACGGCCCTGCACTTGGCCGTTTCG L A N H T G R T A L H L A V S 911 GCTGGCGACGCGGAGATGGCGCGGCACCTGATCGTGTGTGGAGCA A G D A E M A R H L I V C G A 956 TCACCTGTGGCGCGGGATCGCCGGGGCAACACTCCCCTGCACACC S P V A R D R R G N T P L H T 1001 GCCAGCGGCCACGGAGACATCCACATGGTGACCCAGCTTACACGT A S G H G D I H M V T Q L T R 1046 CCCGTCACCGTGGCCGAGGTCATGCACGCCCGCCTGTCCTACGCC P V T V A E V M H A R L S Y A 1091 CCTGCACACACCGCAGGACTCCTGGCCGCTGACCTTACCAACTAC P A H T A G L L A A D L T N Y 1136 GATGGCCAGACGTGTATCCACATTGCGGCGCAGGCTGGACACAAG D G Q T C I H I A A Q A G H K 1181 GAGGTCCTGCAGCATCTCACTTGGTACGGAGCCGACATTAACGCC E V L Q H L T W Y G A D I N A 1226 AAAGAAGGCAAGAGTGGTCGGACGGCACTGCACTACGCCGTAGAG K E G K S G R T A L H Y A V E 1271 GCTCGTGACGCCGACTTGGTGGAATTCCTCACCGAATCCTGCCGA A R D A D L V E F L T E S C R 1316 GCGTCCCTCACCCTGGAGACCTACGCTGGACTTACCCCCTACCAG A S L T L E T Y A G L T P Y Q 1361 CTGGCCTTAGCCAACGGGGCGATGGACCTGGCTCACCAACTGCTG L A L A N G A M D L A H Q L L 1406 AAGCTCGGCGCTCCTGGGGACGCCCTCCCCACCTACCTCACCGCG K L G A P G D A L P T Y L T A 1451 GACGAAGACCTCGACTACGATGAGGTCTCAACTGTGGTTGAGAGC D E D L D Y D E V S T V V E S 1496 TGCAATGGATGGGACAACGTGGATGACCTCAGGATTGGA 1534 C N G W D N V D D L R I G

Rn-NF-kappa-B inhibitor alpha

Mum-NF-kappa-B inhibitor alpha

Bt-NF-kappa-B inhibitor alpha

Hs-NF-kappa-B inhibitor alpha

Mam-NF-kappa-B inhibitor alpha

La-NF-kappa-B inhibitor alpha

Sk-NF-kappa B inhibitor alpha

Pf-NF-kappa B inhibitor alpha

Cg-NF-kappa B inhibitor alpha

Hd-NF-kappa B inhibitor alpha

Mem-NF-kappa B inhibitor alpha

Ll-Cactus

Dm-Cactus

Cf-NF-kappa B inhibitor alpha

Ad-Cactus

Cb-Cactus

Dp-Cactus

Bm-Cactus

Mr-NF-kappa B inhibitor alpha

Pm-Cactus

Lv-Cactus

Fc-Cactus100

100

100

100

100

31

29

100

53

100

100

100

96

99

93

79

64

54

41

0.1

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)

P.monodon 1 ---------------------------------------------MWHIGSAQAQGGVG----ETAVG-- L.vannamei 1 ---------------------------------------------MWHIGSAQAQGGVG----ETAVG-- F.chinensis 1 ---------------------------------------------MWHIGSAQAQGGVG----ETAVG-- M.rosenbergii 1 ---------------------------------------------MWPVGSTHCQGGVINPSSPTTSS-- C.gigas 1 ---------------------------------------------MSNRDFARHQNLDEDDLVEDCAPFG D.melanogaster 1 MPSPTKAAEAATKATATSDCSCSAASVEQRAPSNAANPSSSLATSGKIGGKTQDQTAAINKQKEFAVPNE P.monodon 20 ---SGFAFGSPMSQGRDEGARPKVS-SPTTD--CKSYYDSHN--------DSGFLSGSNLVSSSSLSCED L.vannamei 20 ---SGFAFGSPMSQGRDEGARPKVS-SPTTD--CKSYYDSHN--------DSGFLSGSNLVSSSSLSCEE F.chinensis 20 ---SGFAFGSPMSQGRDEGARPKVS-SPTTD--CKSYYDSHN--------DSGFLSGSNLVSSSSLSCED M.rosenbergii 24 ---S----GLHCALNRDEGARPKTTTSNTTLDTSKSHYDSHH--------DSGFLSGSNLMSSSSIGCDE C.gigas 26 ----G----------------PTEYLKKSAHHFSSDKCDSVC--------DSGVDLRSVESCYSS----- D.melanogaster 71 TSDSGFISGPQSSQIFSEEIVP---DSEEQD---KDQQESAPQKEQPVVLDSGIIDEEEDQEEQ-EKEEE P.monodon 76 NSTMRCKESTSPQEG--------HRSPDPK-----TQGITSVGHLDSGIDISDQLSSLHLAS----ST-- L.vannamei 76 TSSMRCKESTSPQEG--------HRSPDPK-----TQGIPSVGHLDSGIDISDQLSSLHLAS----ST-- F.chinensis 76 NSTMRCKESPSPQEG--------HRSPDPK-----TQGITSVGHLDSGIDISDQLSSLHLAS----ST-- M.rosenbergii 79 PHSPTTATAVSMTGGDGGGGGGPPREDDPRDLKSPTTSATSIGHLDSGIDISEQLSSLHLAS----PT-- C.gigas 63 -------YAGSLPVVAE-------KSPDTQT-------------------IEEKLEGLTLGSQNYPSTEE D.melanogaster 134 HQDTTTATADSMRLK---------HSADTG-----IPQWTVESHLVS---RGEQLNNLGQSS----STQI P.monodon 127 --TSSS--STTSDTPENDTTSSKKVSAPPRRSCLELNEAQLALLQEIFQRDEDGDTQLHVAVMRGFVEVV L.vannamei 127 --TSSS--STTSDTPENDTISPKKVSAPPRRSCLELNEAQLALLQEIFQRDEDGDTQLHVAVMRGFVEVV F.chinensis 127 --TSSS--STTSDTPENDTTSSKKVSAPPRRSCLELNEAQLALLQEIFQRDEDGDTQLHVAVMRGFVEVV M.rosenbergii 143 --TSASPCSTSSEVDLTNATTRKTTSPRPTRSRLGLNQAQVALLQEIFQTDEDGDTQLHVAVMRGFIEVV C.gigas 100 T--------KCSLDDGYISYDRKEVS--CELSHDPTPQISPEVFQLYSQ-DNDGDSQLHMAIINLLVPIA D.melanogaster 183 TGRSKVQSSTASTGNANPSGSGATSSAPPS-SINIMN----AWEQFYQQND-DGDTPLHLACISGSVDVV P.monodon 193 YHITRLLPHQALLDLANHTGRTALHLAVSAGDAEMARHLIVCGASPVARDRRGNTPLHTASGHGDIHMVT L.vannamei 193 YHITRLLPHQALLDLANHTGRTALHLAVSAGDAEMARHLIVCGASPVARDRRGNTPLHTASGHGDIHMVT F.chinensis 193 YHITRLLPHQALLDLANHTGRTALHLAVSAGDAEMARHLIVCGASPVARDRRGNTPLHTASGHGDIHMVT M.rosenbergii 211 YHITRLLPHQAFLDLPNHTGRTALHLAVSTGDSGVARHLVVCGASPVARDRRGNTPLHSASAQGDLRMVT C.gigas 159 LYIIQQAPSRDWLNLPNNMLQTPLHLAVMTRLPQVVKALIDGGADIEARDSKGDTPLHIASREG------ D.melanogaster 247 AALIRMAPHPCLLNIQNDVAQTPLHLAALTAQPNIMRILLLAGAEPTVRDRHGNTALHLSCIAGEKQCVR P.monodon 263 QLTRPVTVAEVMHAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQAGHKEVLQH L.vannamei 263 QLTRPVTVAEVMHAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQAGHKEVLQH F.chinensis 263 QLTRPVTVAEVMHAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQAGHKEVLRH M.rosenbergii 281 QLTRPVTVAEVMNAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQEGHKEILQH C.gigas 223 ----YDDIALILLAP--------------ASTASKRTS----QDLEARNYDGQTCLHLAAENTHLPIIRL D.melanogaster 317 ALTEKFGATEIHEAHRQYGHRSNDKAVSSLSYA------CLPADLEIRNYDGERCVHLAAEAGHIDILRI P.monodon 317 LTWYGADINAKEGKSGRTALHYAVEARDADLVEFLTESCRAS-LTLETYAGLTPYQLALANGAMDLAHQL L.vannamei 317 LTWYGADINAKEGKSGRTALHYAVEARDADLVEFLTESCRAS-LTLETYAGLTPYQLALANGAMDLAHQL F.chinensis 317 LTWYGADINAKEGKSGRTALHYAVEARDADLVEFLTESCRAS-LTLETYAGLTPYQLALANGAMDLAHQL M.rosenbergii 335 LTWYGADINAREGKSGRTALHYAVEARDQALVAFLAESCRAS-LTLETYAGLTPYQLALANGATGIANLL C.gigas 271 LVMSGANLNTQDGKSGKSVIHYAAETGNTLLLDFLLQYSTIN-LHSRTYSGLTAIMLADGRNYHDIVHQL D.melanogaster 381 LVSHGADINAREGKSGRTPLHIAIEGCNEDLANFLLDECEKLNLETATYAGLTAYQFACIMNKSRMQNIL P.monodon 386 LKLG------APGDALPTYLTADEDLDYDEVSTVVESCNGWDNVDDLRIG-------------------- L.vannamei 386 LKLG------APGDALPTYLTADEDLDYDEVSTVVESCNGWDNVDDLRIGGVPVTMAMPVQVNDHQPFNA F.chinensis 386 LKLG------APGDALPTYLTADEDLDYDEVSTVVESCNGWDNVDDLRIGGVPVTMAMPVQVNDHQPFNA M.rosenbergii 404 LSMG------APGDALPTYLTADDDLDYDEVTGPVEISNGWGGMDDLRIGGVPVPMSMPVSVNDSTPFNP C.gigas 340 QKYGGSLENVAFDDSS--------DSEEDMS--------------------------------------- D.melanogaster 451 EKRG------AETVTPP-------DSDYDSS----DIEDLDDTKMYDRFG----DPRYFVSYNGGNPMTV P.monodon ---- L.vannamei 450 DRYF F.chinensis 450 DRYF M.rosenbergii 468 DRYL C.gigas ---- D.melanogaster 500 A---

ResultsPmCactus

F. chinensis: 97% L. vannamei: 99%

ag tcatccagaccaaaatacgggaggaagttactcttattttctctc aagatttctattggtctcggcgttgactgcccatcatgagtgtgt aggcaagtgtgacagaagcctacggtcaaaggaggtgtttgaggc

138 ATGGCTGACCCAATGTTTGTTGCCCAGCGTACTTCCTCCAACCAG M A D P M F V A Q R T S S N Q 183 CTTTCTCTCAACATTAGTGACGTCATTGATATTATTGGCCAAGAT L S L N I S D V I D I I G Q D 228 GACCCTGATTATGCAGCATCAGCCATTTCTGTGTATGGAGGGGTA D P D Y A A S A I S V Y G G V 273 CAGATGGGAGGAGGCGGAGGAGGAGGGGCTGCAAGTGAAGCAAAC Q M G G G G G G G A A S E A N 318 TTTTCTACCATATCCTCTGCGAGTAGACCTGACCCCGATCTGGAG F S T I S S A S R P D P D L E 363 AGCAAACGTAAAGCCTATGTACGTATTATAGAACAGCCCCAGGCT S K R K A Y V R I I E Q P Q A 408 AAGGCACTCCGTTTTCGTTATATTTGTGAAGGACGTTCAGCTGGA K A L R F R Y I C E G R S A G 453 TCAATACCAGGAGTTCGAAGTACAACAGAAAATAAAACTTACCCA S I P G V R S T T E N K T Y P 498 GCTATTCAGGTGGTAGGATACAAAGGACCTGCTGTGGTGGTGGTG A I Q V V G Y K G P A V V V V 543 TCCTGTGTCACTGTTGACCCACCTTACCGACCCCACCCACACAAC S C V T V D P P Y R P H P H N 588 TTGGTGGGGAAGGAAGGATGCAAAAAAGGCGTGTGTACAATGACC L V G K E G C K K G V C T M T 633 ATTAACAATGACTCCATGCAGTGTGTCTTTAGTAACTTGGGAATC I N N D S M Q C V F S N L G I 678 CAGTGTGTCAAAAAACGTGATGTGGAAGATGCCCTCAAATTACGA Q C V K K R D V E D A L K L R 723 GAGGAGATTAGAGTGGACCCTTTCCAGACTGGGTTTTCTCATCGT E E I R V D P F Q T G F S H R 768 AACCAGCCACAGAGCATTGACCTAAATGCTCTGAGATTGTGTTTT N Q P Q S I D L N A L R L C F 813 CAAGTATTCCTAGAGGGATCAGAAAAGGGGAAATTTACCTTCCCT Q V F L E G S E K G K F T F P 858 TTAAAACCACAGGTTTCAGATCCAATTTACGACAAAAAGGCCACA L K P Q V S D P I Y D K K A T 903 TCAGATCTGATTATCTGCAAGCTCAGTGACTGCACGAGCAGTGTG S D L I I C K L S D C T S S V 948 GCTGGTGGCAAAGAGATTATTCTCCTCTGTGATAAAGTTACTAAA A G G K E I I L L C D K V T K 993 GAGGACATTCATGTCCGCTTCTATGAGGTCAAAGATGGAATGATA E D I H V R F Y E V K D G M I 1038 GAATGGGAAGCCTTTGGTGACTTCCAAGCTTCTGATGTCCACAAG E W E A F G D F Q A S D V H K 1083 CAAGTGGCCATTTCGTTTAAGACTCCCAGATATAAAACTCTTGAG Q V A I S F K T P R Y K T L E 1128 ATAGAGAACCCCATAAAGGTGTACGTGC 1155 I E N P I K V Y V

5’ UTR

Rel Homology Domain (RHD)

82-252aa

Ig-like, plexins and

transcription factors (IPT)

domain257-339aa

PmDorsalResults

PmDorsal cDNA partial sequence – 1155bp (339aa)

PmDorsal protein domain topology

Lv-Dorsal

Ec-Dorsal

Pm-Dorsal

Fc-Dorsal

Ae-Dorsal

Cq-Dorsal

Tc-Dorsal

Dm-Dorsal

Cb-Dorsal

Cf-Dorsal

Nv-Dorsal

Hs-Dorsal

99

62

100

99

95

91

68

57

100

0.05

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)  

P.monodon 1 MADPMFVAQRTSSNQLSLNISDVIDIIGQDDPDYAASAISVYGGVQMGGGGGGG-AASEA L.vannamei 1 ----MFVAQRTSSNQLSLHISDVIDIIGQDDPDYAASAISVYGGVQMGGGGGGGGAASEA F.chinensis 1 --------------------------------------------MTPGGGG----AASEA E.sinensis 1 MADPMFVARR-SNNQVTLNISDVIDIIGQDAPECAASDMSVYGGLQIGGDGAG--AASEA D.melanogaster 1 ----MFPNQ---NNGAAP---------GQGP--------AVDG--QQSLNYNGLPAQQQQ P.monodon 60 NFSTISSASRPDPDLESKRKAYVRIIEQPQAKALRFRYICEGRSAGSIPGVRSTTENKTY L.vannamei 57 NFSTISSASRPDPDLESKRKAYVRIIEQPQAKALRFRYICEGRSAGSIPGVRSTTENKTY F.chinensis 13 NFSTISSASRPDPDLESKRKAYVRIIEQPQAKALRFRYICEGRSAGSIPGVRSTTENKTY E.sinensis 58 NFSTLTSLSRTDPDMESKRKAYVRILEQPQPKALRFRYICEGRSAGSIPGVSSTAENKTY D.melanogaster 35 QLAQSTKNVRKKP--------YVKITEQPAGKALRFRYECEGRSAGSIPGVNSTPENKTY P.monodon 120 PAIQVVGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG L.vannamei 117 PAIQVVGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG F.chinensis 73 PAIQVVGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG E.sinensis 118 PAIQVMGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG D.melanogaster 87 PTIEIVGYKGRAVVVVSCVTKDTPYRPHPHNLVGKEGCKKGVCTLEINSETMRAVFSNLG P.monodon 180 IQCVKKRDVEDALKLREEIRVDPFQTGFSHRNQPQSIDLNALRLCFQVFLEGSEKGKFTF L.vannamei 177 IQCVKKRDVEDALKLREEIRVDPFQTGFSHRNQPQSIDLNALRLCFQVFLEGSEKGKFTF F.chinensis 133 IQCVKKRDVEDALKLREEIRVDPFQTGFSHRNQPQSIDLNALRLCFQVFLEGSEKGKFTF E.sinensis 178 IQCVKKRDVDDALKLREEIRVDPFQTGFSHRSQTQSIDLNALRLCFQVFLEGPEKGKFTF D.melanogaster 147 IQCVKKKDIEAALKAREEIRVDPFKTGFSHRFQPSSIDLNSVRLCFQVFMESEQKGRFTS P.monodon 240 PLKPQVSDPIYDKKATSDLIICKLSDCTSSVAGGKEIILLCDKVTKEDIHVRFYEVKDGM L.vannamei 237 PLKPQVSDPIYDKKATSDLIICKLSDCTSSVAGGKEIILLCDKVTKEDIHVRFYEVKDGM F.chinensis 193 PLKPQVSDPIYDKKATSDLIICKLSDCASSVAGGKEIILLCDKVTKEDIHVRFYEVKDGM E.sinensis 238 PLKAVVSHPIYDKKATSDLIICKLSDCTSSVAGGKEIILLCDKVTKEDIQVRFYEEKDGL D.melanogaster 207 PLPPVVSEPIFDKKAMSDLVICRLCSCSATVFGNTQIILLCEKVAKEDISVRFFEEKNGQ P.monodon 300 IEWEAFGDFQASDVHKQVAISFKTPRYKTLEIENPIKVYV-------------------- L.vannamei 297 IEWEAFGDFQASDVHKQVAISFKTPRYKTLEIENPIKVYVQLLRPPDKSTSEPRPFQYLP F.chinensis 253 IEWEAFGDFQASDVHKQVAISFKTPRYKTLEIENPIKVYVQLLRPSDKSTSEPRPFQYLP E.sinensis 298 LEWEGYGDFQASDVHKQVAISFKTPRYKSLEVENPVKVYVQLLRPSDKSMSEPRPFQYLP D.melanogaster 267 SVWEAFGDFQHTDVHKQTAITFKTPRYHTLDITEPAKVFIQLRRPSDGVTSEALPFEYVP

Results

PmDorsal

F. chinensis: 99% L. vannamei: 99%

Discussion

DORSAL Litopenaeus vannamei

Huang et al. 2010

Fenneropenaeus chinensis

Li et al. 2010

Penaeus monodon

Full-length sequence 2206bp 1568bp 1155bp

ORF 1200bp 1071bp 1018bp

Putative protein 400aa 357aa 339aa

RHD 79-249aa 35-205aa 82-252aa

IPT 254-355aa 210-311aa 257-339aa

GenBank (Acc. No.) FJ998202.1 EU815056.1

CACTUS Litopenaeus vannamei

Li et al. 2012

Fenneropenaeus chinensis

Wang et al. 2013

Penaeus monodon

Full-length sequence 2829bp 1926bp 1534bp

ORF 1359bp 1359bp 1018bp

Putative protein 453aa 453aa 463aa

ANK 1 175-204aa 175-204aa 209-238aa

ANK 2 211-240aa 211-240aa 245-274aa

ANK 3 244-273aa 244-273aa 278-307aa

ANK 4 297-326aa 297-326aa 331-360aa

ANK 5 331-361aa 331-361aa 365-395aa

ANK 6 365-398aa 365-398aa 399-432aa

GenBank (Acc. No.) JX014314.1 JQ693681

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

Results and Discussion

Results and DiscussionTissue-level expression

Maximum expression

Minimum expression

CACTUS PmCactus Midgut, muscle

Cephalic ganglion, hindgut

LvCactus Heart , muscle

Hepatopancreas Li et al. 2012

FcCactus Muscle Testis, eye stalk Wang et al. 2013

DORSAL PmDorsal Lymphoid organ

Hepatopancreas

LvDorsal Gill, haemocytes (lymphoid organ-not reported)

Muscle, heart, hepatopancreas

Huang et al. 2010

FcDorsal Haemocyte, lymphoid organ

Hepatopancreas, eye stalk

Li et al. 2010

Gene expression in post larvae (PL-18)-Immersion challenge with WSSV and Vibrio

harveyi

Results

Gene expression in gill-Immersion challenge with

WSSV and Vibrio harveyi

Results

Gene expression - WSSV injection challenge

Vibrio harveyi

Results

Gene expression - Vibrio harveyi injection challenge

Expression in response to WSSV and LPS in Haemocytes in vitro

Prim

ary h

aem

ocyte

cu

lture

ResultsW

SS

VL

PS

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 of LvCactus could be noticed in the haemocytes of L. vannamei in reponse to V. parahemolyticus challenge, except at 12 h and 48 hpi (Li et al., 2012).

Progressive reduction in the expression level of LvCactus could be noticed after WSSV challenge (Li et al., 2012).

Wang et al. (2013) noticed that in response to V. alginolyticus, expression level of FcCactus 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

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.

Discussion

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.

Conclusion

National Fund for Basic, Strategic and Frontier Application Research in Agriculture (NFBSFARA),

ICAR

Director, Central Institute of Fisheries Education (CIFE), Mumbai

Acknowledgements

Thank You

• Arts, J.A., Cornelissen, F.H., Cijsouw, T., Hermsen, T., Savelkoul, H.F., Stet, R.J., 2007. Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon. Fish Shellfish Immunol.23, 504–13.

• Ausvet Animal Health Services, 1997. Scientific Review of Prawn Diseases. A report to the Australian Quarantine and Inspection Service. Australian Quarantine and Inspection Service, Canberra.

• Bachere, E., 2000. Shrimp immunity and disease control. Aquaculture. 191, 3–11.

• Deepika, A., Sreedharan, K., Paria, A., Makesh, M., Rajendran, K.V., 2014. Toll pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: Evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes. Fish Shellfish Immunol.41, 441-454.

• Jiravanichpaisal, P., Puanglarp, N., Petkon, S., Donnuea, S., Soderhall, I., Soderhall, K., 2007.Expression of immune-related genes in larval stages of the giant tiger shrimp, Penaeus monodon.Fish Shellfish Immunol. 23, 815–824.

• Jose, S., Mohandas, A., Philip, R., Bright Singh, I.S., 2010.Primary haemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays. J. Invertebr. Pathol. 105, 312–321.

• Li, F., Wang, D., Li, S., Yan, H., Zhang, J., Xiang, J., 2010. A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge. Dev. Comp.Immunol.34, 874–883.

• Livak, K.J., Schmittgen, T.D., 2001.Analysis of relative gene expression data using Real-

• Rajendran, K.V., Vijayan, K.K., Santiago, T.C., Krol, R.M., 1999.Experimental host range and histopathology of white spot syndrome virus (WSSV) infection in shrimp, prawns, crabs and lobsters from India. J. Fish Dis. 22,133–191.

• Watthanasurorot, A., Söderhäll, K., Jiravanichpaisal, P., 2012. A mammalian like interleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediator in intestinal innate immunity of black tiger shrimp Penaeus monodon. Biochem. Biophys. Res. Commun. 417(1), 623–629.

References