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Chapter 7 their expression will probably not cause damage to the crustacean host. In addition, evidence is accumulating that these compounds may have anti- viral properties. Compounds that effect up-regulation of genes coding for specific antimicrobial peptides could be explored by screening currently available immunostimulants. However, so far hardly any information is available on the expression of antimicrobial peptide (AMP) genes in shrimps in response to the administration of immunostimulants/probiotics and WSSV infection. Therefore, the present work was aimed at characterizing AMP genes in two important penaeid shrimps viz. P.monodon and F. indicus and studying the differential expression of AMP genes in response to immunostimulant/probiotic administration pre- and post-challenge WSSV. The objectives of the study were: � Molecular characterization and phylogenetic analysis of AMPs in Penaeus monodon and Fenneropenaeus indicus � Time-course of AMP gene expression in response to WSSV challenge in Penaeus monodon � Tissue-wise expression profile of AMP genes in Penaeus monodon � Expression profile of AMP genes in response to the administration of immunostimulants in Penaeus monodon pre- and post-challenge WSSV � Expression profile of AMP genes in response to the administration of probiotic bacteria in Penaeus monodon pre- and post-challenge WSSV Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 390
Salient findings Chapter 7 � From P. monodon, seven AMP genes belonging to ALF, crustin and penaeidin family viz. ALF-1, ALF-2, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 could be characterized at molecular level. � From F. indicus, five AMP genes belonging to ALF, crustin and penaeidin family viz. ALF-1, ALF-2, Fi-crustin, penaeidin-5 and Fi- penaeidin and could be characterized at molecular level. � The phylogenetic analysis performed for the AMPs of P. monodon and F. indicus revealed that the AMPs of shrimps were evolutionarily closely related to AMPs of other decapods species. The molecular phylogenetic tree implied that each AMP family possesses a same ancestral origin, which has subsequently diverged at different phases of evolution. � Control genes viz. 18S rRNA, β-actin and elongation factor were also characterized from P. monodon and F. indicus. � All the nucleotide sequences and the deduced amino acid sequences were submitted to GenBank under the following accession numbers: � P. monodon � ALF-1 - GU732817 � ALF-2 - HM588914 � Crustin-1 - GQ334395 � Crustin-2 - FJ535568 � Crustin-3 - GQ334396 � Penaeidin-3 - GU732819 � Penaeidin-5 - GQ334397 Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 391
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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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Chapter 3 Fig.3.5. Multiple alignme
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Chapter 3 Fig.3.8. Nucleotide and a
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Chapter 3 Fig.3.12. Multiple alignm
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Chapter 3 Fig.3.14. A bootstrapped
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SHRIMPS KIND CRAB LOBSTERS CRABS Ch
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Chapter 3 Fig. 3.28. Multiple align
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PENAEIDIN-2 Chapter 3 PENAEIDIN- 3
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Fig.3.31. Nucleotide and amino acid
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Chapter 3 Fig.3.37 Nucleotide and a
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4.1. Introduction Chapter 4 The aqu
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Chapter 4 appearance and have major
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Chapter 4 the tissues has recently
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Chapter 4 with the ability of shrim
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Chapter 4 with a low dose of WSSV b
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Chapter 4 molecules they are likely
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Chapter 4 for understanding gene ex
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Chapter 4 4.3.2. Expression profile
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Expression profile of endonuclease
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Chapter 4 bacterial and fungal infe
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Chapter 4 measuring TSV and YHV loa
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Chapter 4 Of particular interest he
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Chapter 4 large distribution and ab
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Chapter 4 WSSV genes viz. endonucle
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Table 4.2. Primers used for the stu
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 F
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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CHAPTER-5 Expression Profile of Ant
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Chapter 5 1994). The emergence of b
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Chapter 5 (Bovill et al., 2001). In
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5.2.2. Test diets Chapter 5 Four ex
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5.2.2.4. Preparation of Glucan inco
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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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Page 383 and 384: (A) (B) Fig. 6.1. Experimental diet
Page 385 and 386: (A) (B) (C) Fig. 6.3. Expression pr
Page 387 and 388: (A) (B) (C) C B M BM C B M BM Fig.
Page 389 and 390: (A) (B) (C) Fig. 6.7. Expression pr
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Page 407 and 408: (A) (B) G M HP HR I Chapter 6 Fig.
Page 409 and 410: Chapter 6 Fig. 6.27. Post challenge
Page 411: 7.1. Summary Chapter 7 Tropical shr
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Page 417 and 418: Chapter 7 application in shrimp cul
Page 419 and 420: References Aboudy, Y., Mendelson, E
Page 421 and 422: References Anggraeni, M.S., Owens,
Page 423 and 424: References Bals, R., Wang, X., Zasl
Page 425 and 426: References derived from the N-termi
Page 427 and 428: References Breukink, E., de Kruijff
Page 429 and 430: References Burgents, J.E., Burnett,
Page 431 and 432: References 1,3-β-D-glucan-binding
Page 433 and 434: References Chen, J.Y., Chuang, H.,
Page 435 and 436: References Cole, A.M., Hong, T., Bo
Page 437 and 438: References Dathe, M., Wieprecht, T.
Page 439 and 440: References Diamond, G., Zasloff, M.
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Page 445 and 446: References Gennaro, R., Zanetti, M.
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Page 449 and 450: References Harwig, S.S., Swiderek,
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Page 459 and 460: References Kang, J.H., Shin, S.Y.,
Page 461 and 462: 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 antimicrobial peptide fr
Page 479 and 480:
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 Roth, B.L., Poot, M., Yu
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References Sathish, S., Musthaq, S.
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References a proline-arginine-rich
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References Smith, V.J., Fernandes,
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References Song, Y.L., Cheng, W., W
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References experimental system. In:
Page 499 and 500:
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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References Vives, R.R., Imberty, A.
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References Wang, Y.C., Chang, P.S.,
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References Williams, M.J., Rodrigue
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References Wu, W., Zong, R., Xu, J.
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References Yodmuang, S., Tirasophon
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References Zhan, W.B., Wang, Y.H. 1
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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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