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Chapter 4 shrimp cells and its complete internalization (Yi et al. 2004). This protein seems to participate in viral attachment to shrimp cells and plays a key role during the initial step of cell infection (van Hulten et al., 2001; Wu et al., 2005). Latency genes show maximal rate of transcription following viral DNA synthesis, and are involved in the persistence of the virus within a host cell (Flint et al., 2000). Their function is to keep a low number of viruses and inactivating host genes, until the optimal conditions of pH, salinity and temperature are present. Viral immediate early genes are expressed immediately after primary infection, or as a result of the reactivation of a virus. This class of genes is defined experimentally by their ability to produce transcripts even in the presence of inhibitors of protein synthesis (Liu et al., 2005). The expression of viral immediate early genes depends on the host cell machinery and occurs independently of any viral de novo protein synthesis, which means that the immediate early genes are especially important in determining host range (Friesen, 1997). WSSV temporal regulatory genes do not require viral proteins to be transcribed, and are expressed using the host molecular machinery in the first hours after infection (Liu et al., 2005). In the present study, all WSSV genes were found to amplify within 48 hrs of WSSV challenge (Fig. 4.12 to 4.19). Pattern of expression was almost similar for all the eight WSSV genes studied, except that four of the WSSV genes viz. DNA polymerase, endonuclease, protein kinase and VP28 genes could be detected from 24 hours of WSSV challenge onwards; whereas other four WSSV genes viz. immediate early gene, latency related gene, ribonucleotide reductase and thymidine kinase were detected only from 48 hours of challenge. The results showed that only a small amount of transcripts of DNA polymerase, ribonucleotide reductase and VP28 could be detected during early hours of WSSV challenge and that the expression level remained unchanged until post-challenge day 4. Whereas, in case of other Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 263
Chapter 4 WSSV genes viz. endonuclease, immediate early gene, latency related gene, protein kinase, and thymidine kinase higher levels of transcripts could be detected. However, in these samples also the expression levels remained more or less similar until post-challenge day 4. After that, the amount of all WSSV gene transcripts dramatically increased from 5 th day post-challenge and continued to increase during the experimental period. The expression kinetics of the various WSSV gene transcripts were thus found to be quite similar in that all genes were found to be expressed during 1 st or 2 nd day post-challenge onwards and were found to be expressed at maximum level at the later stages of WSSV infection i.e. from 5 th day of post-challenge onwards. This in turn shows that WSSV infection has reached its peak from 5 th day onwards. The present results are in agreement with previous works by Liu et al. (2005) in which, expression profile of DNA polymerase and VP28 was studied for 36 hours post-challenge WSSV. Lin et al. (2002) has studied the time-course of ribonucleotide reductase for 84 hrs of post-WSSV challenge. Lu et al. (2005) have studied the time-course analysis of immediate early gene for 48 hrs post-challenge WSSV injection. The results showed that the WSSV gene were transcribed as early as 2 hours post-challenge and after that, the amount transcripts continued to increase until the end of the analysis. Similar results have been reported by earlier researchers for VP35 gene (Chen et al., 2002a), DNA polymerase gene (Chen et al., 2002b), protein kinase gene (Liu et al., 2001), ribonucleotide reductase gene (Tsai et al., 2000a) and thymidine kinase gene (Tsai et al., 2000b). Viral replication could be the predominant process in the haemocytes of WSSV-infected shrimp, allowing them to overcome the expression of shrimp immune related genes (Chen et al., 2008b). In the present study also, viral replication was found to overcome immune response in the experimental animals during late hours of WSSV infection, except for ALF and crustin-3. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 264
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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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Chapter 3 random coiled structure t
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3.3.1.3.5. Phylogenetic analysis of
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Chapter 3 case has been reported in
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Chapter 3 Multiple alignments were
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Chapter 3 negative bacteria. In shr
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Chapter 3 indicated a random coiled
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Chapter 3 horseshoe crab big defens
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Table 3.4. BLASTn analysis of ALF-2
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Table.3.8. BLASTn analysis of Fi-pe
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Chapter 3 Fig.3.2. Nucleotide and a
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Page 235 and 236: Chapter 3 Fig.3.12. Multiple alignm
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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
Page 261 and 262: Chapter 4 the tissues has recently
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Page 267 and 268: Chapter 4 molecules they are likely
Page 269 and 270: Chapter 4 for understanding gene ex
Page 271 and 272: Chapter 4 4.3.2. Expression profile
Page 273 and 274: Expression profile of endonuclease
Page 275 and 276: Chapter 4 bacterial and fungal infe
Page 277 and 278: Chapter 4 measuring TSV and YHV loa
Page 279 and 280: Chapter 4 Of particular interest he
Page 281: Chapter 4 large distribution and ab
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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
Page 307 and 308: Chapter 5 (Bovill et al., 2001). In
Page 309 and 310: 5.2.2. Test diets Chapter 5 Four ex
Page 311 and 312: 5.2.2.4. Preparation of Glucan inco
Page 313 and 314: Chapter 5 genes (latency related ge
Page 315 and 316: Chapter 5 On WSSV challenge, crusti
Page 317 and 318: Chapter 5 5.3.4. Expression profile
Page 319 and 320: 5.3.4.5. Expression profile of pena
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Page 323 and 324: Chapter 5 For the present study two
Page 325 and 326: Chapter 5 ameobocytes of two marine
Page 327 and 328: Chapter 5 variation in expression o
Page 329 and 330: Chapter 5 It has been previously sh
Page 331 and 332: 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 Li, L., Wang, J.X., Zhao
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References Liu, C.H., Cheng, W., Ku
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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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