title: Identification of prohormones and pituitary neuropeptides in the African cichlid, Astatotilapia burtoni.
abstract: Cichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni, a well-studied member of the diverse African cichlid clade.
  
  
  
  Utilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages.
  
  
  
  We characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.
authors.affiliation: Department of Biology, Stanford University, Stanford, CA, 94305, USA.|Present address: Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, 02138, USA.
authors.forename: Caroline K
authors.initials: CK
authors.lastname: Hu
authors.affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Southey
authors.affiliation: Department of Chemistry and the Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Romanova
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA.
authors.forename: Karen P
authors.initials: KP
authors.lastname: Maruska
authors.affiliation: Department of Chemistry and the Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
authors.forename: Jonathan V
authors.initials: JV
authors.lastname: Sweedler
authors.affiliation: Department of Biology, Stanford University, Stanford, CA, 94305, USA. rfernald@stanford.edu.
authors.forename: Russell D
authors.initials: RD
authors.lastname: Fernald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023681:Cichlids / Q000235:genetics*; D019295:Computational Biology; D019143:Evolution, Molecular; D020440:Gene Duplication; D020869:Gene Expression Profiling; D016678:Genome; D023281:Genomics / Q000379:methods; D006728:Hormones / Q000737:chemistry / Q000235:genetics*; D005810:Multigene Family; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics*; D010902:Pituitary Gland / Q000378:metabolism
chemical_list: D006728:Hormones; D009479:Neuropeptides
keywords: Astatotilapia burtoni; Cichlid; Mass spectrometry; Neuropeptide; Prohormone