Skip to main content
SpringerLink
Log in

Proteomic Analyses of Courtship Pheromones in the Redback Salamander, Plethodon cinereus

  • Published:
Journal of Chemical Ecology Aims and scope Submit manuscript

Abstract

The evolutionary success of plethodontid salamanders for ~100 MY is due partly to the use of courtship pheromones that regulate female receptivity. In ~90 % of plethodontid species, males deliver pheromones by “scratching” a female’s dorsum, where pheromones diffuse transdermally into the bloodstream. However, in a single clade, representing ~10 % of Plethodon spp., males apply pheromones to the female’s nares for olfactory delivery. Molecular studies have identified three major pheromone families: Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). SPF and PMF genes are relatively ancient and found in all plethodontid species; however, PRF is found exclusively in the genus Plethodon – which includes species with transdermal, olfactory, and intermediate delivery behaviors. While previous proteomic analyses suggested PRF and PMF are dominant in slapping species and SPF is dominant in non-Plethodon scratching species, it was unclear how protein expression of different pheromone components may vary across delivery modes within Plethodon. Therefore, the aim of this study was to proteomically characterize the pheromones of a key scratching species in this evolutionary transition, Plethodon cinereus. Using mass spectrometry-based techniques, our data support the functional replacement of SPF by PRF in Plethodon spp. and an increase in PMF gene duplication events in both lineage-dependent and delivery-dependent manners. Novel glycosylation was observed on P. cinereus PRFs, which may modulate the metabolism and/or mechanism of action for PRF in scratching species. Cumulatively, these molecular data suggest that the replacement of pheromone components (e.g., SPF by PRF) preceded the evolutionary transition of the functional complex from transdermal to olfactory delivery.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Arnold SJ (1972) The evolution of courtship behavior in salamanders. University of Michigan, Ann Arbor

    Google Scholar 

  • Arnold SJ (1983) Morphology, performance and fitness. Am Zool 23:347–361

    Google Scholar 

  • Arnold SJ (2005) The ultimate causes of phenotypic integration: lost in translation. Evolution 59:2059–2061

    Google Scholar 

  • Arshad N, Ballal S, Visweswariah SS (2013) Site-specific N-linked glycosylation of receptor guanylyl cyclase C regulates ligand binding, ligand-mediated activation and interaction with vesicular integral membrane protein 36, VIP36. J Biol Chem 288:3907–3917

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cheverud JM (1984) Quantitative genetics and developmental constraints on evolution by selection. J Theor Bio 110:155–171

    Article  CAS  Google Scholar 

  • Chouinard AJ, Wilburn DB, Houck LD, Feldhoff RC (2013) Individual variation in pheromone isoform ratios of the red-legged salamander, Plethodon shermani. In: East ML, Dehnhard M (eds) Chemical signals in vertebrates 12. Springer, New York, pp 99–115

    Chapter  Google Scholar 

  • Date-Ito A, Ohara H, Ichikawa M, Mori Y, Hagino-Yamagishi K (2008) Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system. Chem Senses 33:339–346

    Article  CAS  PubMed  Google Scholar 

  • Dulac C, Torello AT (2003) Molecular detection of pheromone signals in mammals: from genes to behaviour. Nat Rev Neurosci 4:551–562

    Article  CAS  PubMed  Google Scholar 

  • Dyal LA (2006) Novel courtship behaviors in three small eastern Plethodon species. J Herpetol 40:55–65

    Article  Google Scholar 

  • Escoubas P, Diochot S, Corzo G (2000) Structure and pharmacology of spider venom neurotoxins. Biochimie 82:893–907

    Article  CAS  PubMed  Google Scholar 

  • Espiritu DJD, Watkins M, Dia-Monje V, Cartier GE, Cruz LJ, Olivera BM (2001) Venomous cone snails: molecular phylogeny and the generation of toxin diversity. Toxicon 39:1899–1916

    Article  CAS  PubMed  Google Scholar 

  • Estes S, Arnold SJ (2007) Resolving the paradox of stasis: models with stabilizing selection explain evolutionary divergence on all timescales. Amer Nat 169:227–244

    Article  Google Scholar 

  • Evans JP (2002) The molecular basis of sperm-oocyte membrane interactions during mammalian fertilization. Hum Repro Update 8:297–311

    Article  CAS  Google Scholar 

  • Feldhoff RC, Rollmann SM, Houck LD (1999) Chemical analyses of courtship pheromones in a Plethodontid salamander. In: Johnston RE, Műller-Schwarze D, Sorensen P (eds) Advances in chemical signals in vertebrates. Kluwer Academic/Plenum, New York, pp 117–125

    Chapter  Google Scholar 

  • Fry BG (2005) From genome to “venome”: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res 15:403–420

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Haga S, Hattori T, Sato T, Sato K, Matsuda S, Kobayakawa R, Sakano H, Yoshihara Y, Kikusui T, Touhara K (2010) The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor. Nature 466:118–122

    Article  CAS  PubMed  Google Scholar 

  • Hagino-Yamagishi K, Moriya K, Kubo H, Wakabayashi Y, Isobe N, Saito S, Ichikawa M, Yazaki K (2004) Expression of vomeronasal receptor genes in Xenopus laevis. J of Comp Neurol 472:246–256

    Article  CAS  Google Scholar 

  • Herrada G, Dulac C (1997) A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution. Cell 90:763–773

    Article  CAS  PubMed  Google Scholar 

  • Highton R, Hastings AP, Palmer C, Watts R, Hass CA, Culver M, Arnold SJ (2012) Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes. Mol Phylog Evol 63:278–290

    Article  CAS  Google Scholar 

  • Houck LD, Arnold SJ (2003) Courtship and mating behavior. In: Sever DM (ed) Phylogeny and reproductive biology of Urodela (Amphibia). Science Publishers, Enfield, New Hampshire, pp 383–424

    Google Scholar 

  • Houck LD, Reagan NL (1990) Male courtship pheromones increase female receptivity in a Plethodontid salamander. An Behav 39:729–734

    Article  Google Scholar 

  • Houck LD, Sever DM (1994) The role of the skin in reproduction and behavior. In: Heatwole H, Barthalamus G (eds) Amphibian biology. Surrey Beatty and Sons, Chipping Norton, Australia, pp 351–381

    Google Scholar 

  • Houck LD, Mead LS, Watts RA, Arnold SJ, Feldhoff PW, Feldhoff RC (2007a) A candidate vertebrate pheromone, SPF, increases female receptivity in a salamander. In: Hurst J, Beynon R, Muller-Schwarze D (eds) Chemical signals in vertebrates 11. Springer, New York, pp 213–221

    Google Scholar 

  • Houck LD, Palmer CA, Watts RA, Arnold SJ, Feldhoff PW, Feldhoff RC (2007b) A new vertebrate courtship pheromone that affects female receptivity in a terrestrial salamander. An Behav 73:315–320

    Article  Google Scholar 

  • Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 9:275–282

    Google Scholar 

  • Kiemnec-Tyburczy KM, Watts RA, Gregg RG, von Borstel D, Arnold SJ (2009) Evolutionary shifts in courtship pheromone composition revealed by EST analysis of plethodontid salamander mental glands. Gene 432:75–81

    Article  CAS  PubMed  Google Scholar 

  • Kiemnec-Tyburczy KM, Woodley SK, Feldhoff PW, Feldhoff RC, Houck LD (2011) Dermal application of courtship pheromones does not influence receptivity in female red-legged salamanders (Plethodon shermani). J Herpetol 45:169–173

    Article  Google Scholar 

  • Kiemnec-Tyburczy KM, Woodley SK, Watts RA, Arnold SJ, Houck LD (2012) Expression of vomeronasal receptors and related signaling molecules in the nasal cavity of a caudate amphibian (Plethodon shermani). Chem Senses 37:335–346

    Article  CAS  PubMed  Google Scholar 

  • Laberge F, Feldhoff RC, Feldhoff PW, Houck LD (2008) Courtship pheromone-induced c-Fos-like immunolabeling in the female salamander brain. Neuroscience 151:329–339

    Article  CAS  PubMed  Google Scholar 

  • Lande R (1980) Sexual dimorphism, sexual selection, and adaptation in polygenic characters. Evolution 34:292–305

    Article  Google Scholar 

  • North SJ, Hitchen PG, Haslam SM, Dell A (2009) Mass spectroscopy in the analysis of N-linked and O-linked glycans. Curr Opin Struct Biol 19:498–506

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Organ JA (1960) Studies on the life history of the salamander, Plethodon welleri. Copeia 1960:34–40

    Article  Google Scholar 

  • Palmer CA, Watts RA, Gregg RG, McCall MA, Houck LD, Highton R, Arnold SJ (2005) Lineage-specific differences in evolutionary mode in a salamander courtship pheromone. Mol Biol Evol 22:2243–2256

    Article  CAS  PubMed  Google Scholar 

  • Palmer CA, Watts RA, Houck LD, Picard AL, Arnold SJ (2007) Evolutionary replacement of components in a salamander pheromone signaling complex: more evidence for phenotypic-molecular decoupling. Evolution 61:202–215

    Article  CAS  PubMed  Google Scholar 

  • Palmer CA, Picard AL, Watts RA, Houck LD, Arnold SJ (2010) Rapid evolution of Plethodontid modulating factor (PMF), a hypervariable salamander courtship pheromone, is driven by positive selection. J Mol Evol 70:427–440

    Article  CAS  PubMed  Google Scholar 

  • Pfister P, Randall J, Montoya-Burgos JI, Rodriguez I (2007) Divergent evolution among teleost V1r receptor genes. PLoS ONE 2:e379

    Article  PubMed Central  PubMed  Google Scholar 

  • Picard AL (2005) Courtship in the zig-zag salamander (Plethodon dorsalis): insights into a transition in pheromone-delivery behavior. Ethology 111:799–809

    Article  Google Scholar 

  • Rollmann SM, Houck LD, Feldhoff RC (1999) Proteinaceous pheromone affecting female receptivity in a terrestrial salamander. Science 285:1907–1909

    Article  CAS  PubMed  Google Scholar 

  • Rozen S, Skaletsky HJ (1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html

  • Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa. Anal Biochem 166:368–379

    Article  PubMed  Google Scholar 

  • Sever DM (1975) Morphology and seasonal variation of the mental hedonic glands of the dwarf salamander, Eurycea quadridigitata (Holbrook). Herpetologica 31:241–251

    Google Scholar 

  • Sola RJ, Griebenow K (2009) Effects of glycosylation on the stability of protein pharmaceuticals. J Pharma Sci 98:1223–1245

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Uyeda JC, Hansen TF, Arnold SJ, Pienaar J (2011) The million-year wait for macroevolutionary bursts. Proc Natl Acad Sci U S A 108:15908–15913

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vieites DR, Min M-S, Wake DB (2007) Rapid diversification and dispersal during periods of global warming by plethodontid salamanders. Proc Natl Acad Sci 104:19903–19907

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vigerust DJ, Shepherd VL (2007) Virus glycosylation: role in virulence and immune interactions. Trends Microbiol 15:211–218

    Article  CAS  PubMed  Google Scholar 

  • Watts RA, Palmer CA, Feldhoff RC, Feldhoff PW, Houck LD, Jones AG, Pfrender ME, Rollmann SM, Arnold SJ (2004) Stabilizing selection on behavior and morphology masks positive selection on the signal in a salamander pheromone signaling complex. Mol Biol Evol 21:1032–1041

    Article  CAS  PubMed  Google Scholar 

  • Welinder KG, Tams JW (1995) Effects of glycosylation on protein folding, stability and solubility. Studies of chemically modified or engineered plant and fungal peroxidases. In: Steffen B. Petersen BS, Sven P (Eds) Progress in Biotechnology, Elsevier, pp 205–210

  • Wilburn DB, Bowen KE, Gregg RG, Cai J, Feldhoff PW, Houck LD, Feldhoff RC (2012) Proteomic and UTR analyses of a rapidly evolving hypervaraible family of vertebrate pheromones. Evolution 66:2227–2239

    Article  CAS  PubMed  Google Scholar 

  • Willaert B, Bossuyt F, Janssenswillen S, Adriaens D, Baggerman G, Matthijs S, Pauwels E, Proost P, Raepsaet A, Schoofs L et al (2013) Frog nuptial pads secrete mating season-specific proteins related to salamander pheromones. J Exp Biol 216:4139–4143

    Article  CAS  PubMed  Google Scholar 

  • Wirsig-Wiechmann CR, Houck LD, Feldhoff PW, Feldhoff RC (2002) Pheromonal activation of vomeronasal neurons in Plethodontid salamanders. Brain Res 952:335–344

    Article  CAS  PubMed  Google Scholar 

  • Wirsig-Wiechmann CR, Houck LD, Wood JM, Feldhoff PW, Feldhoff RC (2006) Male pheromone protein components activate female vomeronasal neurons in the salamander Plethodon shermani. BMC Neurosci 7:26

    Article  PubMed Central  PubMed  Google Scholar 

  • Woodley SK (1994) Plasma androgen levels, spermatogenesis, and secondary sexual characteristics in two species of Plethodontid salamanders with dissociated reproductive patterns. Gen Comp Endocrinol 96:206–214

    Article  CAS  PubMed  Google Scholar 

  • Woodley SK (2007) Sex steroid hormones and sexual dimorphism of chemosensory structures in a terrestrial salamander (Plethodon shermani). Brain Res 1138:95–103

    Article  CAS  PubMed  Google Scholar 

  • Woodley SK (2010) Pheromone communication in amphibians. J Comp Physiol A: Neuroethology, Sensory, Neural, and Behav Physiol 196:713–727

    Article  CAS  Google Scholar 

  • Xu H, Freitas MA (2009) MassMatrix: a database search program for rapid characterization of proteins and peptides from tandem mass spectrometry data. Proteomics 9:1548–1555

    Article  PubMed Central  PubMed  Google Scholar 

  • Yoshinaga S, Sato T, Hirakane M, Esaki K, Hamaguchi T, Haga-Yamanaka S, Tsunoda M, Kimoto H, Shimada I, Touhara K et al (2013) Structure of the mouse sex peptide pheromone ESP1 reveals a molecular basis for specific binding to the class C G-protein-coupled vomeronasal receptor. J Biol Chem 288:16064–16072

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

Plethodon cinereus pheromone extract was generously provided by Drs. Lynne Houck and Stevan Arnold. We thank Kari Doty and Cristin Samuels for assistance in the laboratory, the University of Louisville Biomolecular Mass Spectrometry Core Laboratory (William Pierce, Jian Cai, Ned Smith) for their continued support, and the Highlands and Mountain Lake Biological Stations for continued support of our field research efforts. Funding was provided in part by National Science Foundation (Collaborative) grants IOS-1146899 (RCF) and IOS-1147271 (LDH), a National Science Foundation Graduate Research Fellowship to DBW, and the University of Louisville IRIG program.

Author information

Author notes

  1. Damien B. Wilburn

    Present address: Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Louisville Health Sciences Center, Louisville, KY, 40292, USA

    Damien B. Wilburn, Kathleen E. Bowen, Pamela W. Feldhoff & Richard C. Feldhoff

Authors
  1. Damien B. Wilburn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kathleen E. Bowen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pamela W. Feldhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard C. Feldhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard C. Feldhoff.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

SDS-PAGE analysis of individual RP-HPLC fractions. A representative SDS-PAGE gel comparing individual RP-HPLC fractions (marked by number, see Table 1) to the whole Plethodon cinereus pheromone (Pc) and size standard (L). Notably, the three PRF fractions (11–13) show molecular weights larger than the ~22 kDa predicted from DNA sequences. (GIF 22 kb)

High resolution image (TIFF 484 kb)

Fig. S2

Glycosylation analysis of individual RP-HPLC fractions. Select RP-HPLC fractions were separated by SDS-PAGE and analyzed for glycosylation using both carbohydrate and protein staining. No glycosylation was detected on any of the protein bands. (PNG 1038 kb)

Table S1

(DOCX 22 kb)

Table S2

(DOCX 24 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wilburn, D.B., Bowen, K.E., Feldhoff, P.W. et al. Proteomic Analyses of Courtship Pheromones in the Redback Salamander, Plethodon cinereus . J Chem Ecol 40, 928–939 (2014). https://doi.org/10.1007/s10886-014-0489-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10886-014-0489-y

Keywords

Search

Navigation