title: Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.
abstract: BACKGROUND
  Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP) precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions.
  
  
  RESULTS
  Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France), Vigo (Spain) and Venice (Italy). To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore) and wild (lagoon) Venice mussels, both injected with 10(7) Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC) minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation.
  
  
  CONCLUSIONS
  Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related to geographical origin or Vibrio injection emerged. Whether or not the contact with potential pathogens can increase the amount of AMP transcript variants in mussels requires additional study.
authors.affiliation: Department of Biology, University of Padua, Padova, Italy. umberto.rosani@unipd.it
authors.forename: Umberto
authors.initials: U
authors.lastname: Rosani
authors.forename: Laura
authors.initials: L
authors.lastname: Varotto
authors.forename: Alberta
authors.initials: A
authors.lastname: Rossi
authors.forename: Philippe
authors.initials: P
authors.lastname: Roch
authors.forename: Beatriz
authors.initials: B
authors.lastname: Novoa
authors.forename: Antonio
authors.initials: A
authors.lastname: Figueras
authors.forename: Alberto
authors.initials: A
authors.lastname: Pallavicini
authors.forename: Paola
authors.initials: P
authors.lastname: Venier
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics*; D005060:Europe; D014644:Genetic Variation; D006434:Hemocytes; D006458:Hemolymph; D049878:Mytilus / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D023181:Antimicrobial Cationic Peptides; D012333:RNA, Messenger
references.citation: Autophagy. 2008 Feb;4(2):254-6
references.pmid: 18196967
references.citation: BMC Genomics. 2010 Mar 19;11:187
references.pmid: 20302637
references.citation: Dev Comp Immunol. 2008;32(3):213-26
references.pmid: 17628673
references.citation: Bioinformatics. 2009 May 1;25(9):1189-91
references.pmid: 19151095
references.citation: Nat Med. 2006 Jul;12(7):852-5
references.pmid: 16799556
references.citation: Dis Aquat Organ. 2006 Jun 23;70(3):251-4
references.pmid: 16903237
references.citation: BMC Genomics. 2011 Jan 26;12:69
references.pmid: 21269501
references.citation: Curr Biol. 2008 Oct 14;18(19):1501-7
references.pmid: 18848447
references.citation: Fish Shellfish Immunol. 2007 Apr;22(4):340-50
references.pmid: 16926100
references.citation: Science. 2001 Mar 2;291(5509):1783-5
references.pmid: 11230693
references.citation: Dev Comp Immunol. 2010 Jun;34(6):659-68
references.pmid: 20097222
references.citation: Dev Comp Immunol. 2000 Jun;24(4):381-93
references.pmid: 10736522
references.citation: Methods Mol Biol. 2000;132:365-86
references.pmid: 10547847
references.citation: Genome Res. 2007 Aug;17(8):1195-201
references.pmid: 17600086
references.citation: J Muscle Res Cell Motil. 2008;29(2-5):73-99
references.pmid: 19039672
references.citation: Biochemistry. 2000 Nov 28;39(47):14436-47
references.pmid: 11087396
references.citation: Comp Biochem Physiol A Mol Integr Physiol. 2008 Sep;151(1):108-13
references.pmid: 18593601
references.citation: Mol Immunol. 2009 Jan;46(3):384-92
references.pmid: 19054563
references.citation: Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746-50
references.pmid: 3459152
references.citation: BMC Genomics. 2009 Feb 09;10:72
references.pmid: 19203376
references.citation: Science. 2011 Apr 1;332(6025):43-4
references.pmid: 21454776
references.citation: PLoS One. 2010 Jan 25;5(1):e8875
references.pmid: 20111607
references.citation: Fish Shellfish Immunol. 2011 Mar;30(3):954-63
references.pmid: 21300156
references.citation: PLoS One. 2011 May 05;6(5):e18904
references.pmid: 21573210
references.citation: Anesthesiology. 2010 Jun;112(6):1428-34
references.pmid: 20502117
references.citation: Mol Ecol. 2011 Feb;20(3):517-29
references.pmid: 21199031
references.citation: Eur J Biochem. 1996 Aug 15;240(1):302-6
references.pmid: 8925841
references.citation: Genomics. 2005 Oct;86(4):423-30
references.pmid: 16039093
references.citation: Nat Rev Microbiol. 2007 Sep;5(9):727-40
references.pmid: 17703227
references.citation: Biotechnol Adv. 2011 Sep-Oct;29(5):519-30
references.pmid: 21683779
references.citation: Aquat Toxicol. 2007 Apr 20;82(1):1-14
references.pmid: 17346810
references.citation: Biofouling. 2006;22(3-4):251-9
references.pmid: 17290869
references.citation: Biochimie. 2010 Sep;92(9):1236-41
references.pmid: 20188791
references.citation: PLoS One. 2007 Feb 14;2(2):e197
references.pmid: 17299583
references.citation: Dis Model Mech. 2010 May-Jun;3(5-6):274-9
references.pmid: 20354110
references.citation: RNA Biol. 2010 Mar-Apr;7(2):220-8
references.pmid: 20220309
references.citation: J Invertebr Pathol. 2008 Mar;97(3):197-202
references.pmid: 18076900
references.citation: Dev Comp Immunol. 2009 Sep;33(9):974-9
references.pmid: 19409926
references.citation: Fish Shellfish Immunol. 2010 Apr;28(4):640-8
references.pmid: 20045066
references.citation: Biochem Pharmacol. 1990 Feb 15;39(4):625-9
references.pmid: 1689576
references.citation: Eur J Biochem. 1999 Oct 1;265(1):71-8
references.pmid: 10491159
references.citation: J Cell Sci. 2000 Aug;113 ( Pt 15):2759-69
references.pmid: 10893191
references.citation: Dev Comp Immunol. 2009 Feb;33(2):162-70
references.pmid: 18789353
references.citation: PLoS Pathog. 2008 Jul 18;4(7):e1000105
references.pmid: 18636113
references.citation: PLoS One. 2010 Sep 29;5(9):
references.pmid: 20927379
references.citation: PLoS One. 2011 Feb 11;6(2):e17003
references.pmid: 21347300
references.citation: Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7
references.pmid: 271968
references.citation: Evolution. 2003 May;57(5):1061-77
references.pmid: 12836823
references.citation: Dev Comp Immunol. 2008;32(3):227-38
references.pmid: 17628674
references.citation: J Biol Chem. 2000 Apr 28;275(17):12954-62
references.pmid: 10777596
references.citation: Philos Trans R Soc Lond B Biol Sci. 2009 Jan 12;364(1513):3-14
references.pmid: 18926970
references.citation: PLoS Comput Biol. 2010 Jul 08;6(7):e1000847
references.pmid: 20628614
references.citation: J Immunol. 2008 Dec 15;181(12):8585-94
references.pmid: 19050278
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: J Parasitol. 2010 Feb;96(1):200-5
references.pmid: 19785477
references.citation: Mol Phylogenet Evol. 2008 Oct;49(1):84-91
references.pmid: 18678263
references.citation: Mar Biotechnol (NY). 2011 Oct;13(5):857-67
references.pmid: 21246236
references.citation: Mar Genomics. 2011 Mar;4(1):61-8
references.pmid: 21429466
references.citation: Nucleic Acids Res. 2007;35(19):e130
references.pmid: 17932070
references.citation: Environ Sci Technol. 2010 Nov 1;44(21):8276-83
references.pmid: 20879708
references.citation: Arch Immunol Ther Exp (Warsz). 2010 Feb;58(1):15-25
references.pmid: 20049649
references.citation: FEBS Lett. 2000 Dec 15;486(3):185-90
references.pmid: 11119700
references.citation: J Biol Chem. 1996 Sep 6;271(36):21808-13
references.pmid: 8702979
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417