Deepika Anand, K. Sreedharan,P.P.Suresh Babu, Anutosh Paria, M. Makesh and K. V. Rajendran...
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Transcript of Deepika Anand, K. Sreedharan,P.P.Suresh Babu, Anutosh Paria, M. Makesh and K. V. Rajendran...
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.
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