Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 4 Temporal regulatory genes of WSSV (Liu et al., 2005), epidemics of WSSV (Wu et al., 2001) various host species of WSSV (Jiravanichpaisal et al., 2001; Rodriguez et al., 2003) WSSV pathology (Wang et al., 1995, 2002; Lightner 1996), WSSV transmission (Chang et al., 1996; Lightner et al., 1997) diagnostic methods for WSSV (Liu et al., 2002; Okumura et al., 2004) and control of WSSV (Dupuy et al., 2004; Park et al., 2004; Witteveldt et al., 2004b). Although considerable progress has been made in the characterization of WSSV, little work has been done on the host immune response particularly by haemocytes in response to the viral infection at the molecular level (Soderhall and Cerenius, 1992; Wongpanya et al., 2007). Also, little is known about the molecular mechanisms underlying the WSSV infection and host immune response. To develop effective infection controls, more information about the mode of infection and interaction between the virus and its host is needed. Knowledge of the immune system in shrimp is necessary to develop methods to control and minimize the loss of production due to infectious diseases. Larger numbers of immune-related genes need to be identified and functionally characterized to better understand shrimp immunity. 4.1.1. Antiviral immune response The molecular mechanisms that underlie the crustacean antiviral immune responses are still unknown and only in the early stages of investigation. Recently, studies using different techniques have been carried out on host-WSSV interactions in crustaceans (Lan et al., 2006; Liu et al., 2006; Wang et al., 2006a, 2007b; Zhao et al., 2007). It has been reported that virus-inhibiting proteins could be produced and some genes were up- regulated upon viral infection in crustaceans (Pan et al,. 2000; Rojtinnakorn et al., 2002; Roux et al., 2002; Dhar et al., 2003; He et al., 2005; Pan et al., 2005). Genes induced by viral infections and genes whose expression is associated Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 243
Chapter 4 with the ability of shrimp to survive from viral infections have been widely reported. Crayfish ALF was shown to inhibit WSSV replication both in vivo and in vitro (Liu et al., 2006). The mechanism of this inhibition still needs further investigations. A Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene was up-regulated in WSSV infected shrimp suggesting that shrimp LGBP is an inducible acute-phase protein that may play a critical role in shrimp- WSSV interaction (Roux et al., 2002). PmAV was found in virus resistant shrimp which has a C-type lectin-like domain (CTLD). Recombinant PmAV protein displayed a strong antiviral activity in inhibiting virus-induced cytopathic effect in fish cells invitro. Further experiments showed that PmAV did not bind to the WSSV implying that the antiviral mechanism of this protein was not due to inhibition of the attachment of virus to target host cell (Luo et al., 2003). A β-integrin was found to interact with a WSSV envelope protein VP187 containing the RGD motif. Soluble integrin, integrin-specific antibody and an RGD containing peptide could block the WSSV infection invivo and invitro. Silencing of β-integrin efficiently inhibited the virus infection. These data suggest that this β-integrin may function as a cellular receptor for WSSV infection (Li et al., 2007). A syntenin and its protein partner α-2-macroglobulin co-precipitated with each other and both of them were up-regulated in the acutephase of a WSSV infection (Tonganunt et al., 2005). A chitin-binding protein (PmCBP) interacted with a WSSV067C protein and showed up-regulation at the late stage of WSSV infection (Chen et al. 2007). Actin microfilaments were shown to interact with VP26 in shrimp (Xie and Yang 2005). VP28 of WSSV was suggested to bind to shrimp cells as an attachment protein and could help the virus to enter into the cytoplasm (Yi et al., 2004). Moreover, haemocyanin, the respiratory protein of arthropods and molluscs, was found to delay the infection of WSSV invivo in P. japonicus (Lei et al., 2008). Another C-type-lectin (LvLT) was decreased Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 244
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Molecular Characterization and Gene
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Declaration I hereby do declare tha
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Acknowledgements This work was carr
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My Seniors, Dr. Annies Joseph, Dr.
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Contents Title Page Nos. 1 General
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in response to WSSV challenge 252-2
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α-2M Alpha-2-Macroglobulin ALF Ant
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ppA Prophenoloxidase-Activating Enz
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1.1 Introduction Chapter 1 Aquacult
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Chapter 1 the giant tiger shrimp (P
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Common names: Chapter 1 Giant tiger
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1.3.1. Diseases Chapter 1 Diseases
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Lymphoidal parvo-like virus (LPV) B
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Chapter 1 and protective occlusion
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Chapter 1 small brown masses in the
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1.4.6. Natural epidemics Chapter 1
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Chapter 1 can differentiate WSSV-in
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1.5.1.1. Probiotics Chapter 1 Alter
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Chapter 1 tried in P. monodon, resu
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Chapter 1 The first and essential i
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Chapter 1 1998). The innate immunit
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1.6.1 Haemocytes Chapter 1 The haem
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Chapter 1 In crustaceans, the sheet
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Chapter 1 Theopold et al., 2002). C
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Chapter 1 kill/remove the invading
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Chapter 1 forming complexes with a
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1.7 Antimicrobial Peptides (AMPs) 1
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Chapter 1 the response is very fast
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Chapter 1 Hirsch, 1947). While they
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Chapter 1 AMPs to various degrees u
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1.7.5 Biological activity Chapter 1
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Chapter 1 including the microbes as
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Tachyplesin I Chapter 1 Crustacea P
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Anionic and cationic peptides that
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Anionic and Cationic AMPs with cyst
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Chapter 1 Fig. 1.7. Structural clas
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Chapter 1 Group III: Linear peptide
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Chapter 1 through regular processes
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Chapter 1 ultimately signal to tran
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Chapter 1 divalent polyanionic cati
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Chapter 1 type of transmembrane por
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Chapter 1 other intracellular targe
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Chapter 1 Apidaecin, a short, proli
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Chapter 1 Hultmark, 1987; Gennaro a
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Chapter 1 quite opposite characteri
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Chapter 1 depletion of mitochondria
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Chapter 1 conformations adopted by
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Chapter 1 receptor may be a potenti
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Chapter 1 exposure to their targets
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Chapter 1 generating a rich spectru
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Chapter 1 by enkelytin (Goumon et a
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Lactoferricin Cow Mastitis control
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Chapter 1 trial, in which peptide T
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Chapter 1 a too limited spectrum to
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Chapter 1 number of research groups
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CHAPTER-2 Molecular Characterizatio
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Chapter 2 interaction with negative
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Chapter 2 inhibit the endotoxin or
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Chapter 2 structure be modified to
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Chapter 2 and the region might be l
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Chapter 2 been made of the spectra
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Chapter 2 their discovery and initi
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Chapter 2 terminal pyroglutamic aci
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Chapter 2 One can assume that the p
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Chapter 2 by degranulation into the
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2.2. Materials and Methods 2.2.1. E
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Chapter 2 washed by adding 1 ml 75
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Chapter 2 entry of recombinant DNA
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Chapter 2 and the deduced amino aci
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Chapter 2 Fenneropenaeus, Litopenae
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2.3.1.3. Crustin-1 (GQ334395) Chapt
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2.3.1.3.5. Phylogenetic analysis of
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Chapter 2 sequence also showed high
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Chapter 2 chinensis and F. indicus
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Chapter 2 monodon and Group III of
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Chapter 2 Tiger shrimp (P. monodon)
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Chapter 2 ALF-1 was a partial seque
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Chapter 2 As mentioned before, the
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Chapter 2 Munoz et al., 2004). The
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Table 2.1. Primers used for the stu
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Table 2.4. BLASTn analysis of ALF-2
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Table 2.8. BLASTn analysis of Crust
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Table 2.12. BLASTn analysis of Pena
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Fig.2.5. Agarose gel electrophoreto
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Chapter 2 Fig.2.9. Multiple alignme
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Chapter 2 Fig. 2.12 A bootstrapped
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Chapter 2 Fig. 2.15. Multiple align
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Chapter 2 Fig.2.18. A bootstrapped
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Chapter 2 Fig.2.20. Signal peptide
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Chapter 2 Fig.2.24 Multiple alignme
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Chapter 2 LITOPENAEUS SP. FARFANTEP
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Chapter 2 Fig. 2.28. Nucleotide and
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Chapter 2 LITOPENAEUS SP. FENNEROPE
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Chapter 2 Fig.2.43 A bootstrapped n
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Chapter 2 LITOPENAEUS SP. FENNEROPE
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Chapter 2 Fig.2.51 Signal peptide a
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Chapter 2 FENNEROPENAEUS SP. FARFAN
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CHAPTER-3 Molecular Characterizatio
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Chapter 3 certainly help us to unra
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Chapter 3 3.3.1.1. Anti-lipopolysac
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Chapter 3 was predicted out to be 1
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Page 213 and 214: 3.3.1.3.5. Phylogenetic analysis of
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Page 217 and 218: Chapter 3 Multiple alignments were
Page 219 and 220: Chapter 3 negative bacteria. In shr
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Page 223 and 224: Chapter 3 horseshoe crab big defens
Page 225 and 226: Table 3.4. BLASTn analysis of ALF-2
Page 227 and 228: Table.3.8. BLASTn analysis of Fi-pe
Page 229 and 230: Chapter 3 Fig.3.2. Nucleotide and a
Page 231 and 232: Chapter 3 Fig.3.5. Multiple alignme
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Page 235 and 236: Chapter 3 Fig.3.12. Multiple alignm
Page 237 and 238: Chapter 3 Fig.3.14. A bootstrapped
Page 239 and 240: Chapter 3 Fig.3.17. Signal peptide
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Page 245 and 246: Chapter 3 Fig.3.25. Signal peptide
Page 247 and 248: Chapter 3 Fig. 3.28. Multiple align
Page 249 and 250: PENAEIDIN-2 Chapter 3 PENAEIDIN- 3
Page 251 and 252: Fig.3.31. Nucleotide and amino acid
Page 255 and 256: Chapter 3 Fig.3.37 Nucleotide and a
Page 257 and 258: 4.1. Introduction Chapter 4 The aqu
Page 259 and 260: Chapter 4 appearance and have major
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Page 267 and 268: Chapter 4 molecules they are likely
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Page 271 and 272: Chapter 4 4.3.2. Expression profile
Page 273 and 274: Expression profile of endonuclease
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Page 277 and 278: Chapter 4 measuring TSV and YHV loa
Page 279 and 280: Chapter 4 Of particular interest he
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Page 283 and 284: Chapter 4 WSSV genes viz. endonucle
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Page 303 and 304: CHAPTER-5 Expression Profile of Ant
Page 305 and 306: Chapter 5 1994). The emergence of b
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Page 309 and 310: 5.2.2. Test diets Chapter 5 Four ex
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Chapter 5 genes (latency related ge
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Chapter 5 On WSSV challenge, crusti
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Chapter 5 5.3.4. Expression profile
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5.3.4.5. Expression profile of pena
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Chapter 5 must firstly rest on a so
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Chapter 5 For the present study two
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Chapter 5 ameobocytes of two marine
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Chapter 5 variation in expression o
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Chapter 5 It has been previously sh
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Chapter 5 manifested in terms of gr
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(A) (B) (C) C CHY CSY CHG CSG C CHY
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(A) (B) -VE CTRL +VE CTRL CHG CSG C
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(A) PRE-CHALLENGE (B) POST-CHALLENG
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(A) PRE-CHALLENGE (B) (C) (D) CONTR
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(C) (D) (A) PRE-CHALLENGE (B) CONTR
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(A) (B) G M HP HR I Chapter 5 Fig.
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Chapter 5 Fig. 5.26. Post challenge
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6.1. Introduction Chapter 6 Prophyl
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Chapter 6 production of inhibitory
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6.2. Materials and methods 6.2.1. E
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Chapter 6 tissue cDNA was performed
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Chapter 6 gene was supported by Bac
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Chapter 6 treated group of shrimps.
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Chapter 6 the gene was found to be
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Chapter 6 found to support minimum
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Chapter 6 is induced upon bacterial
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Chapter 6 all the target genes, exc
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Chapter 6 Detailed tissue-wise anal
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Chapter 6 Gills and intestine was f
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(A) (B) Fig. 6.1. Experimental diet
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(A) (B) (C) Fig. 6.3. Expression pr
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(A) (B) (C) C B M BM C B M BM Fig.
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(A) (B) (C) Fig. 6.7. Expression pr
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(A) (B) (C) Chapter 6 Fig. 6.9. Exp
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(A) (B) -VE CTRL +VE CTRL B M BM Ch
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(A) (C) (D) PRE-CHALLENGE POST-CHAL
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(A) (B) G M HP HR I Chapter 6 Fig.
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Chapter 6 Fig. 6.27. Post challenge
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7.1. Summary Chapter 7 Tropical shr
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Salient findings Chapter 7 � From
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Chapter 7 � Tissue-wise expressio
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Chapter 7 application in shrimp cul
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References Aboudy, Y., Mendelson, E
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References Anggraeni, M.S., Owens,
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References Bals, R., Wang, X., Zasl
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References derived from the N-termi
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References Breukink, E., de Kruijff
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References Burgents, J.E., Burnett,
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References 1,3-β-D-glucan-binding
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References Chen, J.Y., Chuang, H.,
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References Cole, A.M., Hong, T., Bo
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References Dathe, M., Wieprecht, T.
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References Diamond, G., Zasloff, M.
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References Faber, C., Stallmann, H.
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References Flint, S.J., Enquist, L.
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References Gennaro, R., Zanetti, M.
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References Gudmundsson, G.H., Lidho
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References Harwig, S.S., Swiderek,
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References Hirakura, Y., Kobayashi,
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References Huang, C.C., Song, Y.L.
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References Itami, T., Takahashi, Y.
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References Jiravanichpaisal, P. Whi
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References Kang, J.H., Shin, S.Y.,
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References Kono, T., Savan, R., Sak
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References rich peptide derived fro
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References Li, L., Wang, J.X., Zhao
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References Liu, C.H., Cheng, W., Ku
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References Lorenzon, S., de Guarrin
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References Marone, M., Mozzetti, S.
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References Medvinsky, A., Dzierzak,
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References Moriarty, D.J.W. 1996. M
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References Otta, S.K., Karunasagar,
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References simplex virus has heptad
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References infected shrimp. Thesis
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References Litopenaeus vannamei cha
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References Sathish, S., Musthaq, S.
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References a proline-arginine-rich
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References Song, Y.L., Cheng, W., W
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References experimental system. In:
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References the black tiger shrimp P
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References Tapay, L.M., Cesar, E.,
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References Touhy, K.M., Probert, H.
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References van Hulten, M.C., Reijns
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Penaeus monodon anti-lipopolysaccha
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Penaeus monodon crustin-like antimi
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Penaeus monodon crustin-like antimi
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Penaeus monodon penaeidin-like anti
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Penaeus monodon elongation factor m
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Fenneropenaeus indicus anti-lipopol
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Fenneropenaeus indicus penaeidin-li
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Fenneropenaeus indicus beta-actin m
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PUBLICATIONS
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Short sequence report Molecular cha
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Fig. 2. Multiple alignment of nucle
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220 The phylogenetic relationships
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Table 1 Rearing conditions and wate
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5 ′ cttgtggttgacaatggctccg3
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Expression of WSSV genes in the hae
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Fig. 4. Expression profile of crust
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S.P. Antony et al. / Immunobiology
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