Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 6 Six animals from each group were sampled after 14 days of probiotic supplementation for the pre-challenge data. On the 15 th day all the groups were orally challenged with WSSV by feeding WSSV infected frozen prawn tissue at the rate of 1g / animal. Thereafter the animals were maintained on their respective diets. After 48 h five animals each from all the groups were sampled for the post-challenge data and rest of the shrimps were maintained on respective diets for observing mortality, if any. During sampling only those shrimps in the intermoult stage were evaluated in the study. Survival in each treatment group was recorded daily for a period of seven days with dead animals removed promptly. Mortality by WSSV infection was confirmed by checking the characteristic white spots on the carapace of infected shrimps. 6.2.4. Haemolymph and other tissues collection Haemolymph and tissues including gill, muscle, heart, hepatopancreas and intestine were collected for total RNA isolation as described in section 5.2.4. 6.2.5. Total RNA isolation and reverse transcription Total RNA isolation and first strand cDNA synthesis were performed as described in section 2.2.3. to 2.2.6. 6.2.6. Semi-quantitative RT-PCR analysis of gene expression Expression of the target genes when supplemented with different probiotic bacteria pre- and post-challenge WSSV was determined by semi- quantitative RT-PCR analysis using 18S rRNA, β-actin and elongation factor (ELF) as the internal control (Marone et al., 2001) as described in section 4.2.6. From the three different probiotics screened, the best one was selected based on AMP gene expression, intensity of WSSV infection as envisaged from the WSSV gene expression and post-challenge survival data. The best performed probiotic group along with control group was subjected to detailed tissue-wise expression profile analysis. PCR amplifications of the Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 343
Chapter 6 tissue cDNA was performed for six AMP genes (ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5) and two WSSV genes (latency related gene and VP28) in a 25 µl reaction volume as described in section 5.2.6. 6.2.7. Statistical analysis Statistical significance of the post challenge survival data of the control group and probiotic administered groups were determined by One- way ANOVA and Duncan’s multiple comparison of the means using the software SPSS 10.0. A probability (p) value of less than 0.05 was considered significant. 6.3. Results 6.3.1. Expression profile of control genes in the haemocytes of P. monodon in response to the administration of probiotic bacteria pre- and post-challenge WSSV Expression profile of three control genes viz. 18S rRNA (Fig. 6.2), β- actin (Fig. 6.3) and ELF (Fig. 6.4) were analyzed. ELF was found to be the best reference gene. 6.3.2. Expression profile of AMP genes in the haemocytes of P. monodon in response to the administration of probiotic bacteria pre- and post-challenge WSSV Expression profile of all AMP genes viz. ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 in the haemocytes of P. monodon were found to vary on administration of the probiotic bacteria both pre- and post- challenge WSSV (Fig. 6.5–6.10). WSSV infection was found to modulate AMP gene expression in haemocytes of both the control and probiotic administered group of animals. 6.3.2.1. Expression profile of ALF Noticeable variation in the expression of ALF could be observed on administration of probiotic bacteria. ALF was found to be constitutively Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 344
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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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Page 385 and 386: (A) (B) (C) Fig. 6.3. Expression pr
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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 and 412: 7.1. Summary Chapter 7 Tropical shr
Page 413 and 414: 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
Page 429 and 430:
References Burgents, J.E., Burnett,
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References 1,3-β-D-glucan-binding
Page 433 and 434:
References Chen, J.Y., Chuang, H.,
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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.
Page 441 and 442:
References Faber, C., Stallmann, H.
Page 443 and 444:
References Flint, S.J., Enquist, L.
Page 445 and 446:
References Gennaro, R., Zanetti, M.
Page 447 and 448:
References Gudmundsson, G.H., Lidho
Page 449 and 450:
References Harwig, S.S., Swiderek,
Page 451 and 452:
References Hirakura, Y., Kobayashi,
Page 453 and 454:
References Huang, C.C., Song, Y.L.
Page 455 and 456:
References Itami, T., Takahashi, Y.
Page 457 and 458:
References Jiravanichpaisal, P. Whi
Page 459 and 460:
References Kang, J.H., Shin, S.Y.,
Page 461 and 462:
References Kono, T., Savan, R., Sak
Page 463 and 464:
References rich peptide derived fro
Page 465 and 466:
References Li, L., Wang, J.X., Zhao
Page 467 and 468:
References Liu, C.H., Cheng, W., Ku
Page 469 and 470:
References Lorenzon, S., de Guarrin
Page 471 and 472:
References Marone, M., Mozzetti, S.
Page 473 and 474:
References Medvinsky, A., Dzierzak,
Page 475 and 476:
References Moriarty, D.J.W. 1996. M
Page 477 and 478:
References antimicrobial peptide fr
Page 479 and 480:
References Otta, S.K., Karunasagar,
Page 481 and 482:
References simplex virus has heptad
Page 483 and 484:
References infected shrimp. Thesis
Page 485 and 486:
References Litopenaeus vannamei cha
Page 487 and 488:
References Roth, B.L., Poot, M., Yu
Page 489 and 490:
References Sathish, S., Musthaq, S.
Page 491 and 492:
References a proline-arginine-rich
Page 493 and 494:
References Smith, V.J., Fernandes,
Page 495 and 496:
References Song, Y.L., Cheng, W., W
Page 497 and 498:
References experimental system. In:
Page 499 and 500:
References the black tiger shrimp P
Page 501 and 502:
References Tapay, L.M., Cesar, E.,
Page 503 and 504:
References Touhy, K.M., Probert, H.
Page 505 and 506:
References van Hulten, M.C., Reijns
Page 507 and 508:
References Vives, R.R., Imberty, A.
Page 509 and 510:
References Wang, Y.C., Chang, P.S.,
Page 511 and 512:
References Williams, M.J., Rodrigue
Page 513 and 514:
References Wu, W., Zong, R., Xu, J.
Page 515 and 516:
References Yodmuang, S., Tirasophon
Page 517 and 518:
References Zhan, W.B., Wang, Y.H. 1
Page 519 and 520:
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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