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Chapter 6 On challenge with WSSV, a general down-regulation of the penaeidin- 3 genes could be noticed in various target tissues. Penaeidin-3 transcripts could be found only in the gills and intestine of the WSSV challenged shrimps, intestine supporting maximum expression of the gene. But in case of Bacillus treated group, the gene was found to be up-regulated considerably in all the tissues, except heart. Gill was found to support maximum expression of the gene, followed by intestine, muscle and hepatopancreas (Fig. 6. 23 B & D). 6.3.4.6. Expression profile of penaeidin-5 Very low level of penaeidin-5 expression could be observed in the various tissues of gills, muscle and heart of the control group of shrimps. And no transcripts were detected in the hepatopancreas and intestine prior to WSSV challenge. Whereas in the Bacillus administered group of shrimps, up-regulation of the gene was noticed, with gill and muscle supporting maximum expression followed by intestine (Fig. 6. 24 A & C). On challenge with WSSV, up-regulation of the penaeidin-5 gene could be noticed in the various target tissues of the control group of shrimps. Maximum expression was supported by the gills and intestine. Bacillus treated group also supported maximum up-regulation of the penaeidin-5 gene, and maximum expression was found in the intestine followed by gills and muscle (Fig. 6. 24 B & D). 6.3.5. Expression profile of WSSV genes in various tissues of P. monodon in response to the administration of probiotic Bacillus Since, all the WSSV genes studied in the haemocytes of Bacillus treated group, was found to follow a similar pattern of expression; only two WSSV genes were considered for studying the tissue-wise expression profile. In the control group both WSSV genes analyzed viz. the latency related gene (Fig. 6.25) and VP 28 gene (Fig. 6.26), gave amplifications and followed a similar pattern of expression for the various target tissues analyzed. Muscle was Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 351
Chapter 6 found to support minimum expression the WSSV genes. Whereas, in the case of Bacillus administered group of shrimps, none of the target tissues were found to be infected with WSSV. 6.3.6. Post-challenge survival When compared to that of the control group of animals, probiotic bacteria administered groups gave higher survival rate on WSSV challenge. Among the probiotic treated groups, Bacillus administered group of shrimps showed significantly higher survival rate (73%), followed by Micrococcus (73%) and Bacillus + Micrococcus (56%) (Fig. 6.27). 6.4. Discussion Although the exact mechanism is still not yet completely understood, several probiotic preparations are being applied in shrimp culture systems, to induce and build up protection against a wide range of diseases. In order to develop effective intervention strategies for disease control in shrimp culture, a scientific basis for the health modulators in shrimp is required. Several studies have been carried out on the enhancement of the immune response with respect to the application of probiotics. But there are hardly any studies on the effect of these probiotic products on the gene expression of AMPs. Also, the expression profile of immune genes during WSSV infection is also limited, especially AMPs. The response of AMP expression to bacterial challenge is supposed to be enigmatic and often does not follow the pattern expected for immune genes (Gross et al., 2001; Rojtinnakorn et al., 2002; Munoz et al., 2004; Lorgeril et al., 2005; Beale et al., 2008). Hence the present study was undertaken to study the expression profile of AMP genes in response to probiotic bacterial administration and WSSV challenge and thereby to analyze the mechanism of action of probionts in terms of AMP gene expression and to determine the antiviral properties of the major AMP families of shrimps. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 352
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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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Page 325 and 326: Chapter 5 ameobocytes of two marine
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Page 331 and 332: Chapter 5 manifested in terms of gr
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Page 355 and 356: (A) (B) G M HP HR I Chapter 5 Fig.
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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 and 380: Chapter 6 Detailed tissue-wise anal
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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
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,
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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 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 simplex virus has heptad
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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 Smith, V.J., Fernandes,
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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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