Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 6 On WSSV challenge, a noticeable down-regulation of the penaeidin-3 gene could be found in the control group of shrimps. However, considerable up-regulation of penaeidin-3 gene could be observed in case of probiotic administered groups. Maximum up-regulation of the penaeidin-3 gene was supported by Bacillus administered group of shrimps, followed by Bacillus + Micrococcus and Micrococcus (Fig.6.9 B & C). 6.3.2.6. Expression profile of penaeidin-5 Though much variation in the expression of penaeidin-5 could not be observed on administration of probiotic bacteria, slight up-regulation of the gene could be noticed in the probiotic administered groups (Fig.6.10 A & C). On WSSV challenge, penaeidin-5 gene was found to be down- regulated in the control group of shrimps. However, when compared to the control group, the gene was found to be up-regulated in all the probiotic administered groups. Bacillus was found to support maximum up-regulation of the penaedin-5 gene post-challenge WSSV, followed by Micrococcus and Bacillus + Micrococcus (Fig.6.10 B & C). 6.3.3. Expression of WSSV genes in haemocytes of P. monodon in response to the administration of probiotic bacteria All WSSV gene transcripts could be detected in the haemocytes of the control group of shrimps confirming WSSV infection in this group. All the WSSV genes tested followed more or less similar pattern of expression for the various experimental groups analyzed, especially in case of the control group of shrimps. WSSV infection could be confirmed by the expression of DNA polymerase (Fig.6.11), endonuclease (Fig.6.12), immediate early gene (Fig.6.13), latency related gene (Fig.6.14), protein kinase (Fig.6.15), ribonucleotide reductase (Fig.6.16), thymidine kinase (Fig.6.17) and VP28 (Fig.6.18) genes of WSSV in the control group of shrimps. However, probiotic administered group was found to be less infected by WSSV, as indicated by the expression profile of WSSV genes in these Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 347
Chapter 6 treated group of shrimps. Micrococcus administered group of animals also gave positive amplifications for the WSSV genes viz. immediate early gene, ribonucleotide reductase and VP28 gene. Whereas, Bacillus + Micrococcus gave positive amplification only for immediate early gene of WSSV. Hence, these two probiotic administered group were found to be less infected with WSSV when compared to that of control group of shrimps. Interestingly, the Bacillus administered group of shrimps did not give any amplification for the WSSV gene studied. Since Bacillus administered group was found to perform best in terms of AMP gene expression and WSSV gene expression, a detailed tissue-wise expression profile was performed for the above group of shrimps. 6.3.4. Expression profile of AMP genes in various tissues of P. monodon in response to the administration of probiotic Bacillus pre- and post-challenge WSSV AMP gene transcripts could be detected in all the target tissues at varying levels in the animals. In general gills and intestine were found to have maximum transcripts followed by muscle, hepatopancreas and heart. However, the expression profile of each was found to vary for the various tissue targets (Fig. 6.19 – 6.24). 6.3.4.1. Expression profile of ALF Transcripts of ALF could be detected at high levels in the gills, intestine, hepatopancreas and muscle of the control group of P. monodon. In case of the control group of shrimps, gills and intestine supported maximum expression of the ALF gene followed by hepatopancreas and muscle. The ALF gene was found to be up-regulated on Bacillus administration pre- challenge WSSV, except in the hepatopancreas. Maximum up-regulation of the gene was found to be in the muscle of the Bacillus administered group (Fig. 6. 19 A & C). Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 348
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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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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
Page 415 and 416: Chapter 7 � Tissue-wise expressio
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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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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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