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
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 Fisheries
July 20th-22nd , 2015
Brisbane,Australia
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 Gramnegative bacterial infections and virus infection
JAK/STAT pathway - antiviral defense
(Li and Xiang 2013)
Components of Toll pathway in shrimp
Introduction
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) receptorassociated 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
Cactus (IkB homolog) and Dorsal (NF-kappa B homolog) from Invertebrates
Cactus
Dorsal
Pacific oyster, Crassostrea gigas
Pearl oyster, Pinctada fucata
Scallop, Argopecten irradians
Pacific white shrimp, Litopenaeus vannamei
Sea cucumber, Apostichopus japonicus
Abalone, Haliotis discus discus
Chinese shrimp, Fenneropenaeus chinensis
Montagnani et al., 2008
Xiong et al., 2008
Mu et al., 2010
Li et al., 2012
Lu et al., 2013
Kasthuri et al., 2013
Wang et al., 2013
F. chinensis
L. vannamei
Li et al., 2010
Huang et al., 2010
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
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
Materials and methods
cDNA cloning
Primers used in the study
Gene cloning Primers
RNA extraction
(Trizol reagent, Invitrogen, USA)
Quantification
(NanoDrop 2000 spectrophotometer, Thermo Scientific, USA)
PmCactus F1
PmCactus R1
PmDorsal F1
PmDorsal R1
TTCGGATCAACTCAGCTC
CTCCAATCCTGAGGTCATC
TCTGGAGAGCAAACGTAAAG
GCACGTACACCTTTATGG
5’ RACE Primers
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
PmCactus-5’-R1
GAGGAGGTCGTATCGTTTTCGGGCGT
PmCactus-5’- NR1
AGGTAGTAGAGGAGGCCAGGTGAAG
PmDorsal-5’-R1
GACACAGGACACCACCACCACAGCA
PmDorsal-5’-NR1
CCTACCACCTGAATAGCTGGGTAAGT
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
Materials and methods
WSSV /Vibrio harveyi injection Challenge
WSSV inoculum
preparation
(Rajendran et al., 1999)
Clarification
Testing virulence
In vitro – cold
2X L-15 cell
culture medium
(Jose et al., 2010)
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
Collection of Haemolymph
aseptically
Centrifugation
(400g for 3 min)
Haemolymph
withdrawn from
ventral sinus
Collection
Haemocytes by
centrifugation (400g
for 3 min)
v
v
Filtration
After 3 h of exposure, transferred
to fresh seawater having the
same salinity
WSSV inoculum Copy number: 1.3 x
103 copies/µL; V.
harveyi- 106 cfu/mL
v
v
Homogenization
in chilled PBS
• 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
Surface sterilisation
with 70% alcohol
v
v
Gill and pleopod
Tissue samples of
WSSV-infected
P. monodon,
Injection challenge
with WSSV/ V. harveyi
Immersion challenge
with WSSV/ V. harveyi
Preparation of primary haemocyte
culture and exposure to WSSV
Three replicates used
for each time point
Tissues collected at 2,
6, 12, 24, 48, and 72 h
time points postinjection
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
RNA isolation and cDNA synthesis
Materials and methods
Real-time quantitative PCR to estimate mRNA expression
Template: 100 ng cDNA
Real-time PCR primers
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.
EF-1α-F
GGTGCTGGACAAGCTGAAGGC
EF-1α-R
CGTTCCGGTGATCATGTTCTTGATG
PmCactusQF
TTCGGATCAACTCAGCTC
PmCactus QR
CTCCAATCCTGAGGTCATC
PmDorsal QF
GGGTCGAGTGCCAAGTATGC
PmDorsal QR
GCGATCTCGAGCACATTCTTC
Results and discussion
Results
PmCactus
5’ UTR
146
191
236
281
326
371
416
461
506
551
596
641
686
731
776
821
866
911
Ankyrin repeats
(ANK domain)
956
1001
1046
1091
1136
1181
1226
1271
1316
1361
1406
1451
1496
acacacgctc
agtagacttcactgggatcagacgtaggaagtacggcattgagtg
gttatgtttatggtggtgcttgtctagatgaagtggttcagtgtt
ttggtttatagcttggccatgtgtgtgccttagtgagtttactat
ATGCCGATAGAGGTAACTAACGAAATTGTACCCACTGAAGAGTGG
M
P
I
E
V
T
N
E
I
V
P
T
E
E
W
TACTGGGGCGTATTGATCTTGTGTGATAGTCTGTTTACACCTGGA
Y
W
G
V
L
I
L
C
D
S
L
F
T
P
G
CTCTATGCTAAGATGTGGCACATTGGCAGTGCCCAGGCTCAGGGC
L
Y
A
K
M
W
H
I
G
S
A
Q
A
Q
G
GGCGTAGGGGAGACGGCAGTAGGATCGGGGTTTGCCTTCGGCTCC
G
V
G
E
T
A
V
G
S
G
F
A
F
G
S
CCTATGTCACAAGGGAGAGACGAGGGTGCCAGGCCCAAGGTTTCA
P
M
S
Q
G
R
D
E
G
A
R
P
K
V
S
TCCCCTACTACAGACTGCAAATCATACTACGACTCTCACAACGAC
S
P
T
T
D
C
K
S
Y
Y
D
S
H
N
D
TCGGGTTTCCTGTCTGGTTCAAACCTTGTGTCCTCCTCGAGTCTA
S
G
F
L
S
G
S
N
L
V
S
S
S
S
L
AGTTGTGAGGATAACTCCACCATGCGGTGTAAGGAATCCACGTCC
S
C
E
D
N
S
T
M
R
C
K
E
S
T
S
CCTCAGGAGGGCCACAGAAGCCCTGACCCCAAGACTCAGGGCATC
P
Q
E
G
H
R
S
P
D
P
K
T
Q
G
I
ACCTCTGTGGGGCATCTCGATTCCGGCATCGACATTTCGGATCAA
T
S
V
G
H
L
D
S
G
I
D
I
S
D
Q
CTCAGCTCTCTTCACCTGGCCTCCTCTACTACCTCATCCTCGAGC
L
S
S
L
H
L
A
S
S
T
T
S
S
S
S
ACGACGTCAGATACGCCCGAAAACGATACGACCTCCTCCAAGAAG
T
T
S
D
T
P
E
N
D
T
T
S
S
K
K
GTTTCGGCGCCACCACGGCGATCTTGCCTCGAGTTAAACGAAGCT
V
S
A
P
P
R
R
S
C
L
E
L
N
E
A
CAGTTAGCGCTCCTTCAAGAGATATTTCAGCGAGATGAAGACGGA
Q
L
A
L
L
Q
E
I
F
Q
R
D
E
D
G
GACACTCAGCTGCACGTGGCGGTCATGCGTGGCTTCGTCGAGGTG
D
T
Q
L
H
V
A
V
M
R
G
F
V
E
V
GTGTACCACATCACGCGACTCCTCCCCCACCAGGCGCTGCTCGAT
V
Y
H
I
T
R
L
L
P
H
Q
A
L
L
D
CTCGCCAACCACACCGGCAGGACGGCCCTGCACTTGGCCGTTTCG
L
A
N
H
T
G
R
T
A
L
H
L
A
V
S
GCTGGCGACGCGGAGATGGCGCGGCACCTGATCGTGTGTGGAGCA
A
G
D
A
E
M
A
R
H
L
I
V
C
G
A
TCACCTGTGGCGCGGGATCGCCGGGGCAACACTCCCCTGCACACC
S
P
V
A
R
D
R
R
G
N
T
P
L
H
T
GCCAGCGGCCACGGAGACATCCACATGGTGACCCAGCTTACACGT
A
S
G
H
G
D
I
H
M
V
T
Q
L
T
R
CCCGTCACCGTGGCCGAGGTCATGCACGCCCGCCTGTCCTACGCC
P
V
T
V
A
E
V
M
H
A
R
L
S
Y
A
CCTGCACACACCGCAGGACTCCTGGCCGCTGACCTTACCAACTAC
P
A
H
T
A
G
L
L
A
A
D
L
T
N
Y
GATGGCCAGACGTGTATCCACATTGCGGCGCAGGCTGGACACAAG
D
G
Q
T
C
I
H
I
A
A
Q
A
G
H
K
GAGGTCCTGCAGCATCTCACTTGGTACGGAGCCGACATTAACGCC
E
V
L
Q
H
L
T
W
Y
G
A
D
I
N
A
AAAGAAGGCAAGAGTGGTCGGACGGCACTGCACTACGCCGTAGAG
K
E
G
K
S
G
R
T
A
L
H
Y
A
V
E
GCTCGTGACGCCGACTTGGTGGAATTCCTCACCGAATCCTGCCGA
A
R
D
A
D
L
V
E
F
L
T
E
S
C
R
GCGTCCCTCACCCTGGAGACCTACGCTGGACTTACCCCCTACCAG
A
S
L
T
L
E
T
Y
A
G
L
T
P
Y
Q
CTGGCCTTAGCCAACGGGGCGATGGACCTGGCTCACCAACTGCTG
L
A
L
A
N
G
A
M
D
L
A
H
Q
L
L
AAGCTCGGCGCTCCTGGGGACGCCCTCCCCACCTACCTCACCGCG
K
L
G
A
P
G
D
A
L
P
T
Y
L
T
A
GACGAAGACCTCGACTACGATGAGGTCTCAACTGTGGTTGAGAGC
D
E
D
L
D
Y
D
E
V
S
T
V
V
E
S
TGCAATGGATGGGACAACGTGGATGACCTCAGGATTGGA 1534
C
N
G
W
D
N
V
D
D
L
R
I
G
PmCactus cDNA partial
sequence – 1534bp (463aa)
PmCactus protein domain
topology
Results
PmCactus
Rn-NF-kappa-B inhibitor alpha
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
1
1
1
1
1
1
---------------------------------------------MWHIGSAQAQGGVG----ETAVG----------------------------------------------MWHIGSAQAQGGVG----ETAVG----------------------------------------------MWHIGSAQAQGGVG----ETAVG----------------------------------------------MWPVGSTHCQGGVINPSSPTTSS----------------------------------------------MSNRDFARHQNLDEDDLVEDCAPFG
MPSPTKAAEAATKATATSDCSCSAASVEQRAPSNAANPSSSLATSGKIGGKTQDQTAAINKQKEFAVPNE
Mum-NF-kappa-B inhibitor alpha
Bt-NF-kappa-B inhibitor alpha
Hs-NF-kappa-B inhibitor alpha
Mam-NF-kappa-B inhibitor alpha
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
20
20
20
24
26
71
---SGFAFGSPMSQGRDEGARPKVS-SPTTD--CKSYYDSHN--------DSGFLSGSNLVSSSSLSCED
---SGFAFGSPMSQGRDEGARPKVS-SPTTD--CKSYYDSHN--------DSGFLSGSNLVSSSSLSCEE
---SGFAFGSPMSQGRDEGARPKVS-SPTTD--CKSYYDSHN--------DSGFLSGSNLVSSSSLSCED
---S----GLHCALNRDEGARPKTTTSNTTLDTSKSHYDSHH--------DSGFLSGSNLMSSSSIGCDE
----G----------------PTEYLKKSAHHFSSDKCDSVC--------DSGVDLRSVESCYSS----TSDSGFISGPQSSQIFSEEIVP---DSEEQD---KDQQESAPQKEQPVVLDSGIIDEEEDQEEQ-EKEEE
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
76
76
76
79
63
134
NSTMRCKESTSPQEG--------HRSPDPK-----TQGITSVGHLDSGIDISDQLSSLHLAS----ST-TSSMRCKESTSPQEG--------HRSPDPK-----TQGIPSVGHLDSGIDISDQLSSLHLAS----ST-NSTMRCKESPSPQEG--------HRSPDPK-----TQGITSVGHLDSGIDISDQLSSLHLAS----ST-PHSPTTATAVSMTGGDGGGGGGPPREDDPRDLKSPTTSATSIGHLDSGIDISEQLSSLHLAS----PT--------YAGSLPVVAE-------KSPDTQT-------------------IEEKLEGLTLGSQNYPSTEE
HQDTTTATADSMRLK---------HSADTG-----IPQWTVESHLVS---RGEQLNNLGQSS----STQI
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
127
127
127
143
100
183
--TSSS--STTSDTPENDTTSSKKVSAPPRRSCLELNEAQLALLQEIFQRDEDGDTQLHVAVMRGFVEVV
--TSSS--STTSDTPENDTISPKKVSAPPRRSCLELNEAQLALLQEIFQRDEDGDTQLHVAVMRGFVEVV
--TSSS--STTSDTPENDTTSSKKVSAPPRRSCLELNEAQLALLQEIFQRDEDGDTQLHVAVMRGFVEVV
--TSASPCSTSSEVDLTNATTRKTTSPRPTRSRLGLNQAQVALLQEIFQTDEDGDTQLHVAVMRGFIEVV
T--------KCSLDDGYISYDRKEVS--CELSHDPTPQISPEVFQLYSQ-DNDGDSQLHMAIINLLVPIA
TGRSKVQSSTASTGNANPSGSGATSSAPPS-SINIMN----AWEQFYQQND-DGDTPLHLACISGSVDVV
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
193
193
193
211
159
247
YHITRLLPHQALLDLANHTGRTALHLAVSAGDAEMARHLIVCGASPVARDRRGNTPLHTASGHGDIHMVT
YHITRLLPHQALLDLANHTGRTALHLAVSAGDAEMARHLIVCGASPVARDRRGNTPLHTASGHGDIHMVT
YHITRLLPHQALLDLANHTGRTALHLAVSAGDAEMARHLIVCGASPVARDRRGNTPLHTASGHGDIHMVT
YHITRLLPHQAFLDLPNHTGRTALHLAVSTGDSGVARHLVVCGASPVARDRRGNTPLHSASAQGDLRMVT
LYIIQQAPSRDWLNLPNNMLQTPLHLAVMTRLPQVVKALIDGGADIEARDSKGDTPLHIASREG-----AALIRMAPHPCLLNIQNDVAQTPLHLAALTAQPNIMRILLLAGAEPTVRDRHGNTALHLSCIAGEKQCVR
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
263
263
263
281
223
317
QLTRPVTVAEVMHAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQAGHKEVLQH
QLTRPVTVAEVMHAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQAGHKEVLQH
QLTRPVTVAEVMHAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQAGHKEVLRH
QLTRPVTVAEVMNAR--------------LSYAPAHTAGLLAADLT--NYDGQTCIHIAAQEGHKEILQH
----YDDIALILLAP--------------ASTASKRTS----QDLEARNYDGQTCLHLAAENTHLPIIRL
ALTEKFGATEIHEAHRQYGHRSNDKAVSSLSYA------CLPADLEIRNYDGERCVHLAAEAGHIDILRI
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
317
317
317
335
271
381
LTWYGADINAKEGKSGRTALHYAVEARDADLVEFLTESCRAS-LTLETYAGLTPYQLALANGAMDLAHQL
LTWYGADINAKEGKSGRTALHYAVEARDADLVEFLTESCRAS-LTLETYAGLTPYQLALANGAMDLAHQL
LTWYGADINAKEGKSGRTALHYAVEARDADLVEFLTESCRAS-LTLETYAGLTPYQLALANGAMDLAHQL
LTWYGADINAREGKSGRTALHYAVEARDQALVAFLAESCRAS-LTLETYAGLTPYQLALANGATGIANLL
LVMSGANLNTQDGKSGKSVIHYAAETGNTLLLDFLLQYSTIN-LHSRTYSGLTAIMLADGRNYHDIVHQL
LVSHGADINAREGKSGRTPLHIAIEGCNEDLANFLLDECEKLNLETATYAGLTAYQFACIMNKSRMQNIL
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
386
386
386
404
340
451
LKLG------APGDALPTYLTADEDLDYDEVSTVVESCNGWDNVDDLRIG-------------------LKLG------APGDALPTYLTADEDLDYDEVSTVVESCNGWDNVDDLRIGGVPVTMAMPVQVNDHQPFNA
LKLG------APGDALPTYLTADEDLDYDEVSTVVESCNGWDNVDDLRIGGVPVTMAMPVQVNDHQPFNA
LSMG------APGDALPTYLTADDDLDYDEVTGPVEISNGWGGMDDLRIGGVPVPMSMPVSVNDSTPFNP
QKYGGSLENVAFDDSS--------DSEEDMS--------------------------------------EKRG------AETVTPP-------DSDYDSS----DIEDLDDTKMYDRFG----DPRYFVSYNGGNPMTV
P.monodon
L.vannamei
F.chinensis
M.rosenbergii
C.gigas
D.melanogaster
---450 DRYF
450 DRYF
468 DRYL
---500 A---
F. chinensis:
L. vannamei:
97%
99%
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-Cactus
Lv-Litopenaeus
vannamei
(AFO38331);
FcFenneropenaeus
chinensis;
Pm-Penaeus
monodon;Ad-Apis
dorsata
(ACT66871);
MrMacrobrachium 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)
Results
PmDorsal
5’ UTR
Rel Homology
Domain (RHD)
82-252aa
Ig-like, plexins and
transcription factors
(IPT) domain
257-339aa
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
PmDorsal cDNA partial
sequence – 1155bp (339aa)
PmDorsal protein domain
topology
Results
PmDorsal
P.monodon
L.vannamei
F.chinensis
E.sinensis
D.melanogaster
1
1
1
1
1
MADPMFVAQRTSSNQLSLNISDVIDIIGQDDPDYAASAISVYGGVQMGGGGGGG-AASEA
----MFVAQRTSSNQLSLHISDVIDIIGQDDPDYAASAISVYGGVQMGGGGGGGGAASEA
--------------------------------------------MTPGGGG----AASEA
MADPMFVARR-SNNQVTLNISDVIDIIGQDAPECAASDMSVYGGLQIGGDGAG--AASEA
----MFPNQ---NNGAAP---------GQGP--------AVDG--QQSLNYNGLPAQQQQ
P.monodon
L.vannamei
F.chinensis
E.sinensis
D.melanogaster
60
57
13
58
35
NFSTISSASRPDPDLESKRKAYVRIIEQPQAKALRFRYICEGRSAGSIPGVRSTTENKTY
NFSTISSASRPDPDLESKRKAYVRIIEQPQAKALRFRYICEGRSAGSIPGVRSTTENKTY
NFSTISSASRPDPDLESKRKAYVRIIEQPQAKALRFRYICEGRSAGSIPGVRSTTENKTY
NFSTLTSLSRTDPDMESKRKAYVRILEQPQPKALRFRYICEGRSAGSIPGVSSTAENKTY
QLAQSTKNVRKKP--------YVKITEQPAGKALRFRYECEGRSAGSIPGVNSTPENKTY
P.monodon
L.vannamei
F.chinensis
E.sinensis
D.melanogaster
120
117
73
118
87
PAIQVVGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG
PAIQVVGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG
PAIQVVGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG
PAIQVMGYKGPAVVVVSCVTVDPPYRPHPHNLVGKEGCKKGVCTMTINNDSMQCVFSNLG
PTIEIVGYKGRAVVVVSCVTKDTPYRPHPHNLVGKEGCKKGVCTLEINSETMRAVFSNLG
P.monodon
L.vannamei
F.chinensis
E.sinensis
D.melanogaster
180
177
133
178
147
IQCVKKRDVEDALKLREEIRVDPFQTGFSHRNQPQSIDLNALRLCFQVFLEGSEKGKFTF
IQCVKKRDVEDALKLREEIRVDPFQTGFSHRNQPQSIDLNALRLCFQVFLEGSEKGKFTF
IQCVKKRDVEDALKLREEIRVDPFQTGFSHRNQPQSIDLNALRLCFQVFLEGSEKGKFTF
IQCVKKRDVDDALKLREEIRVDPFQTGFSHRSQTQSIDLNALRLCFQVFLEGPEKGKFTF
IQCVKKKDIEAALKAREEIRVDPFKTGFSHRFQPSSIDLNSVRLCFQVFMESEQKGRFTS
P.monodon
L.vannamei
F.chinensis
E.sinensis
D.melanogaster
240
237
193
238
207
PLKPQVSDPIYDKKATSDLIICKLSDCTSSVAGGKEIILLCDKVTKEDIHVRFYEVKDGM
PLKPQVSDPIYDKKATSDLIICKLSDCTSSVAGGKEIILLCDKVTKEDIHVRFYEVKDGM
PLKPQVSDPIYDKKATSDLIICKLSDCASSVAGGKEIILLCDKVTKEDIHVRFYEVKDGM
PLKAVVSHPIYDKKATSDLIICKLSDCTSSVAGGKEIILLCDKVTKEDIQVRFYEEKDGL
PLPPVVSEPIFDKKAMSDLVICRLCSCSATVFGNTQIILLCEKVAKEDISVRFFEEKNGQ
P.monodon
L.vannamei
F.chinensis
E.sinensis
D.melanogaster
300
297
253
298
267
IEWEAFGDFQASDVHKQVAISFKTPRYKTLEIENPIKVYV-------------------IEWEAFGDFQASDVHKQVAISFKTPRYKTLEIENPIKVYVQLLRPPDKSTSEPRPFQYLP
IEWEAFGDFQASDVHKQVAISFKTPRYKTLEIENPIKVYVQLLRPSDKSTSEPRPFQYLP
LEWEGYGDFQASDVHKQVAISFKTPRYKSLEVENPVKVYVQLLRPSDKSMSEPRPFQYLP
SVWEAFGDFQHTDVHKQTAITFKTPRYHTLDITEPAKVFIQLRRPSDGVTSEALPFEYVP
Lv-Dorsal
Ec-Dorsal
Pm-Dorsal
Fc-Dorsal
Ae-Dorsal
F. chinensis:
L. vannamei:
Cq-Dorsal
Tc-Dorsal
Dm-Dorsal
Cb-Dorsal
Cf-Dorsal
Nv-Dorsal
Hs-Dorsal
Lv- Litopenaeus vannamei (ACZ98167); FcFenneropenaeus chinensis (ACJ36225);
Pm-Penaeus
monodon;
Ec-Eriocheir
sinensis
(AHG95994);Tc-Tribolium
castaneum
(NP_001034507);
DmDrosophila melanogaster (AAA28479);AeAedes aegypti (XP_001652840);Cq- Culex
quinquefasciatus
(XP_001844078);CbCerapachys biroi (EZA51108); Nv-Nasonia
vitripennis
(XP_001602675);HsHarpegnathos saltator (XP_011149046); CfCamponotus floridanus (EFN68841)
99%
99%
Discussion
Litopenaeus vannamei
Fenneropenaeus chinensis
Li et al. 2012
Wang et al. 2013
Full-length sequence
2829bp
1926bp
1534bp
ORF
1359bp
1359bp
1018bp
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
CACTUS
Putative protein
Penaeus monodon
Litopenaeus vannamei
Fenneropenaeus chinensis
Huang et al. 2010
Li et al. 2010
Full-length sequence
2206bp
1568bp
1155bp
ORF
1200bp
1071bp
1018bp
400aa
357aa
339aa
RHD
79-249aa
35-205aa
82-252aa
IPT
254-355aa
210-311aa
257-339aa
GenBank (Acc. No.)
FJ998202.1
EU815056.1
DORSAL
Putative protein
Penaeus monodon
Gene expression during developmental stages
Results and Discussion
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
Tissue-level expression
CACTUS
PmCactus
LvCactus
FcCactus
DORSAL
PmDorsal
LvDorsal
FcDorsal
Maximum
expression
Midgut,
muscle
Heart ,
muscle
Muscle
Lymphoid
organ
Gill,
haemocytes
(lymphoid
organ-not
reported)
Haemocyte,
lymphoid
organ
Minimum
expression
Cephalic
ganglion,
hindgut
Hepatopancreas
Testis, eye stalk
Li et al. 2012
Wang et al.
2013
Hepatopancreas
Muscle, heart,
hepatopancreas
Huang et al.
2010
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
Results
WSSV
Primary haemocyte culture
LPS
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 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.
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 Tollpathway 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 Tollpathway, 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.
Acknowledgements
National Fund for Basic, Strategic and Frontier
Application Research in Agriculture (NFBSFARA),
ICAR
Director, Central Institute of Fisheries Education
(CIFE), Mumbai
Thank You
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