Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 6 it is clear that ALF transcription, although tissue specific, occurs in multiple organs and could thereby provide systemic protection against pathogens. In the present study, though a down-regulation of crustins in the tissues could be observed on Bacillus administration pre-challenge WSSV, ALF and penaeidins were found to be up-regulated. This may be because of the fact that, probiotic administration, in effect, mimics a microbial challenge in shrimps and a further WSSV challenge would have resulted in an increasing number of ALF and penaeidin positive haemocytes. Together, the results suggested that haemocytes left the circulation and migrated into shrimp tissues, presumably in order to control and prevent the spreading of the pathogen. A similar observation was seen in the expression of penaeidin in L. vannamei (Munoz et al., 2004; Somboonwiwat et al., 2008). On WSSV challenge, the probiotic (Bacillus) treated group displayed higher up-regulation of all the AMP genes especially crustins and penaeidins. Maximum up-regulation was found to be in the gills and intestine of the treated group of shrimps. Previous studies on penaeidin expression has shown the expression levels of penaeidins to be higher in the haemocytes, gill, heart, and intestine since haemocytes infiltration are more in these tissues (Kang et al., 2004; Liu et al., 2005). Bacillus administration was found to support maximum up-regulation of penaeidins than other AMPs under both pre- and post-challenge conditions. This might be because of the fact that penaeidins are up- regulated in the presence of bacteria (Bachere et al., 2000a; Cuthbertson et al., 2004; Chiou et al., 2005), showing its potential antibacterial activity. Up- regulation of penaeidins on WSSV challenge also seems to be pointing to its possible mode of antiviral defense. This up-regulation was very high on Bacillus administration, which shows that administration of probiotic bacteria could enhance the production of AMPs which in turn could result in better protection against WSSV infection. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 359
Chapter 6 Gills and intestine was found to be the tissues with maximum AMP transcripts. Transcripts observed in the gills and intestine were probably due to the infiltrating haemocytes (Liu et al., 2005). Gill and intestine are important defense barriers of shrimp as they have direct contact with water and food, respectively. Changes in AMP transcript concentration in the tissues shown might be due to the variation in the haemocyte count. Since haemocytes have been proved to be the sole site of production of AMPs in shrimps, expression of AMPs in tissues is believed to be due these infiltrating haemocytes. AMP-expressing haemocytes leave the blood circulation and most of the shrimp tissues via chemotaxis to migrate toward injured tissues. Massive accumulation of AMP-producing haemocytes and free AMPs are then seen around the site of infection and in the muscle tissue, which reveals a striking local anti-microbial response. WSSV which primarily affects the ectodermal and mesodermal tissues could be eliminated by an increased production of immune defense molecules in these tissues. The epithelial lining of gills and tissues are ectodermal in origin. Increased infiltration into these tissues thus in turn fights against WSSV attack delaying the process of infection in prawns. ALF, Crustins and penaeidins have been proved to possess high antibacterial activity (Destoumieux et al., 2001; Smith et al., 2008; Somboonwiwat et al., 2008). The enhanced expression of crustins and penaeidins on administration of probiotic bacteria may be due to its high activity against gram-positive bacteria. This enhanced production of AMPs in turn gives added protection against other pathogenic microbes. Expression of WSSV genes also ensures that the level of WSSV infection is less in the probiotic treated groups when compared to the control group of shrimps (Fig. 6. 11- 6.18; Fig. 6.25 - 6.26). Several modes of action have been proposed for the probiotic bacteria (Antony and Philip, 2008). Action by enhanced production of AMPs could also be considered as one among them. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 360
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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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Page 355 and 356: (A) (B) G M HP HR I Chapter 5 Fig.
Page 357 and 358: Chapter 5 Fig. 5.26. Post challenge
Page 359 and 360: 6.1. Introduction Chapter 6 Prophyl
Page 361 and 362: Chapter 6 production of inhibitory
Page 363 and 364: 6.2. Materials and methods 6.2.1. E
Page 365 and 366: Chapter 6 tissue cDNA was performed
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Page 379: Chapter 6 Detailed tissue-wise anal
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
Page 391 and 392: (A) (B) (C) Chapter 6 Fig. 6.9. Exp
Page 393 and 394: (A) (B) -VE CTRL +VE CTRL B M BM Ch
Page 405 and 406: (A) (C) (D) PRE-CHALLENGE POST-CHAL
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
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,
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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 Kang, J.H., Shin, S.Y.,
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References Kono, T., Savan, R., Sak
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References Liu, C.H., Cheng, W., Ku
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References Otta, S.K., Karunasagar,
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References infected shrimp. Thesis
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References Litopenaeus vannamei cha
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References experimental system. In:
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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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