Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 1 Field methods: Diagnosis of white spot syndrome depends mainly on the demonstration of eosinophilic to basophilic inclusion bodies (IB) in stained fresh squashes or impression smears of ectodermal and mesodermal tissues. Feulgen-positive intranuclear IB may be identified in cuticular epithelial cells and connective tissue cells. The gills and epithelium under the carapace are excised, stained with haemotoxylin and eosin, mounted and then viewed as squash preparations (Flegel and Sriuriairatana, 1993). WSSV infection may be confirmed by the demonstration of rod-shaped, non-occluded virions in the intranuclear IB of affected cells using electron microscopy (Lightner, 1996). There are not many field tests available for the detection of WSSV. A reverse passive latex agglutination assay (RPLA) has been proposed as a useful method for virus detection in the culture shrimp pond (Okumura et al., 2004). Other cost-effective diagnostic assays using fluoresceinated microspheres and latex beads coated with anti-WSSV serum have proved to detect the virus as early as 24 h post infection in shrimps (Sathish et al., 2004). Serological methods: These methods are based on monoclonal or polyclonal antibodies produced against viral antigens or recombinant viral antigens. Monoclonal and polyclonal antibodies produced against VP28 or rVP28 were used to develop several methods including immunofluorescence, immunohistochemistry (Escobedo-Bonilla et al., 2005, 2007), immunoblot assays (You et al., 2002), immunochromatographic test strips (Sithigorngul et al., 2006), enzyme linked immunosorbent assay (ELISA), and western blotting (Nadala et al., 1997; Yoganandhan et al., 2004). Monoclonal antibodies (MAbs) have been produced against WSSV. A 28 kDa envelop protein of WSSV, encoded by the VP28 gene has been extensively used for the preparation of MAbs. These MAbs developed against WSSV are the basis of a number of detection methods. MAbs have also been used to develop an antigen-capture ELISA (Ac-ELISA) test, which Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 16
Chapter 1 can differentiate WSSV-infected shrimp from uninfected shrimp. Antiserum developed against the VP28 is able to neutralize WSSV infections of P. monodon in a concentration- dependent manner on intramuscular injection. Furthermore, the antiserum can detect WSSV in various organs such as the eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages in experimentally infected P. monodon and F. indicus, as early as 12 and 24 h post-infection (Yoganandhan et al., 2004). Molecular methods: These include PCR, in situ hybridization and dot blot hybridization. PCR: These methods are based on primers designed against a specific part of the genome sequence of WSSV. PCR methods include one step PCR (Lightner, 1996; Lo et al., 1996), semi nested PCR (Kiatpathomchai et al., 2001), two step PCR (Tapay et al., 1999; Hossain et al., 2004), quantitative competitive PCR (Tang and Lightner, 2000) and real time PCR (Durand and Lightner, 2002). PCR has been used in cultured and captured crustaceans (Vaseeharan et al., 2006) to estimate the prevalence of the virus in shrimp PL at the time of stocking in shrimp farms, and to identify reservoirs for the virus in shrimp ponds (Thakur et al., 2002). Furthermore, PCR can identify at least four geographic isolates of WSSV from both experimentally and naturally infected shrimp (Tapay et al., 1999). A new protocol called in situ PCR can detect light infections in tissues at early stage of infection (Jian et al., 2005). Another method named loop mediated isothermal amplification (LAMP) is claimed to be more sensitivity than other PCR protocols. It can detect up to 1 femtogram (fg) of virus (Kono et al., 2004). In situ hybridization: This method detects viral DNA in the host tissues through hybridization with a DNA probe (Wongteerasupaya et al., 1996). It is useful to detect infected cells in tissues, but it is less sensitive than PCR and requires histopathology facilities. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 17
Page 1 and 2: Molecular Characterization and Gene
Page 3 and 4: Declaration I hereby do declare tha
Page 5 and 6: Acknowledgements This work was carr
Page 7 and 8: My Seniors, Dr. Annies Joseph, Dr.
Page 9 and 10: Contents Title Page Nos. 1 General
Page 11 and 12: in response to WSSV challenge 252-2
Page 13 and 14: α-2M Alpha-2-Macroglobulin ALF Ant
Page 15 and 16: ppA Prophenoloxidase-Activating Enz
Page 17 and 18: 1.1 Introduction Chapter 1 Aquacult
Page 19 and 20: Chapter 1 the giant tiger shrimp (P
Page 21 and 22: Common names: Chapter 1 Giant tiger
Page 23 and 24: 1.3.1. Diseases Chapter 1 Diseases
Page 25 and 26: Lymphoidal parvo-like virus (LPV) B
Page 27 and 28: Chapter 1 and protective occlusion
Page 29 and 30: Chapter 1 small brown masses in the
Page 31: 1.4.6. Natural epidemics Chapter 1
Page 35 and 36: 1.5.1.1. Probiotics Chapter 1 Alter
Page 37 and 38: Chapter 1 tried in P. monodon, resu
Page 39 and 40: Chapter 1 The first and essential i
Page 41 and 42: Chapter 1 1998). The innate immunit
Page 43 and 44: 1.6.1 Haemocytes Chapter 1 The haem
Page 45 and 46: Chapter 1 In crustaceans, the sheet
Page 47 and 48: Chapter 1 Theopold et al., 2002). C
Page 49 and 50: Chapter 1 kill/remove the invading
Page 51 and 52: Chapter 1 forming complexes with a
Page 53 and 54: 1.7 Antimicrobial Peptides (AMPs) 1
Page 55 and 56: Chapter 1 the response is very fast
Page 57 and 58: Chapter 1 Hirsch, 1947). While they
Page 59 and 60: Chapter 1 AMPs to various degrees u
Page 61 and 62: 1.7.5 Biological activity Chapter 1
Page 63 and 64: Chapter 1 including the microbes as
Page 65 and 66: Tachyplesin I Chapter 1 Crustacea P
Page 67 and 68: Anionic and cationic peptides that
Page 69 and 70: Anionic and Cationic AMPs with cyst
Page 71 and 72: Chapter 1 Fig. 1.7. Structural clas
Page 73 and 74: Chapter 1 Group III: Linear peptide
Page 75 and 76: Chapter 1 through regular processes
Page 77 and 78: Chapter 1 ultimately signal to tran
Page 79 and 80: Chapter 1 divalent polyanionic cati
Page 81 and 82: 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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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 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 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 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 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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