Chemical Senses Advance Access originally published online on June 5, 2008
Chemical Senses 2008 33(7):623-631; doi:10.1093/chemse/bjn027
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A Recombinant Courtship Pheromone Affects Sexual Receptivity in a Plethodontid Salamander
1 Department of Zoology, 3029 Cordley Hall, Oregon State University, Corvallis, OR 97331, USA 2 Department of Biochemistry, University of Louisville, Louisville, KY 40292, USA 3 Department of Environmental Health Sciences, University of California–Los Angeles, Los Angeles, CA 90095, USA
Correspondence to be sent to: Lynne D. Houck, Department of Zoology, 3029 Cordley Hall, Oregon State University, Corvallis, OR 97331, USA. e-mail: houckl{at}science.oregonstate.edu
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Abstract |
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Pheromones are important chemical signals for many vertebrates, particularly during reproductive interactions. In the terrestrial salamander Plethodon shermani, a male delivers proteinaceous pheromones to the female as part of their ritualistic courtship behavior. These pheromones increase the female's receptivity to mating, as shown by a reduction in courtship duration. One pheromone component in particular is plethodontid receptivity factor (PRF), a 22-kDa protein with multiple isoforms. This protein alone can act as a courtship pheromone that causes the female to be more receptive. We used a bacterial expression system to synthesize a single recombinant isoform of PRF. The recombinant protein was identical to the native PRF, based on mass spectrometry, circular dichroism spectra, and a behavioral bioassay that tested the effects of recombinant PRF (rPRF) on female receptivity (21% reduction in courtship duration). The rPRF appears to mimic the activity of a mixture of PRF isoforms, as well as a mixture of multiple different proteins that comprise the male courtship gland extract. Pheromones that are peptides have been characterized for some vertebrates; to date, however, rPRF is one of only 2 synthesized vertebrate proteins to retain full biological activity.
Key words: Plethodon shermani, plethodontid receptivity factor, recombinant protein pheromone, red-legged salamander, vertebrate pheromones
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Introduction |
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Pheromones are chemical signals that are released from an individual and elicit a specific response in a member of the same species (Karlson and Lüscher 1959
). In vertebrates, pheromone signals can elicit a variety of responses in both females and males, including mate attraction, maternal behavior, and reproductive cycle modulation (Meredith 1983; Jemiolo et al. 1986; Kikuyama et al. 1995; Novotny, Jemiolo, et al. 1999; Novotny, Ma, et al. 1999; Wabnitz et al. 1999; Nakada et al. 2007). A behavioral response to pheromone signals often can be observed and quantified, but actually isolating and identifying the specific pheromone compounds that elicit the response is a continuing challenge in chemical ecology. Much of the research on vertebrate pheromones has focused on mammalian communication (Brennan and Zufall 2006). To date, however, only 7 mammalian pheromones have been chemically synthesized and then validated with an experiment that reveals full behavioral response to the synthetic pheromone. The number of characterized pheromones is even lower in amphibians. Prior to this study, pheromones were characterized for one anuran (the magnificent tree frog, Litoria splendida; Wabnitz et al. 1999) and 2 congeneric species of newts (Cynops spp., family Salamandridae; Kikuyama and Toyoda 1999; Yamamoto et al. 2000; Nakada et al. 2007). These amphibian pheromones are peptides, and each has been synthetically produced and the synthetic product behaviorally validated (Table 1).
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We now report the characterization and validation of a male pheromone present in a multispecies group of lungless salamanders within the genus Plethodon (family Plethodontidae). Plethodontid salamanders are nocturnal animals that rely heavily on chemical communication to locate prey items, as well as to identify potential mates. The pheromone we characterized is not used for mate location, however, but is only delivered after courtship interactions between a male–female pair already have begun. A plethodontid pair will engage in a stereotyped sequence of courtship interactions that culminate in sperm transfer via a spermatophore (a gelatinous base supporting an apical sperm cap). One of the courtship actions that precedes sperm transfer is the "tail-straddling walk," during which the female follows behind the male while straddling his tail and with her chin on his tail (a behavior that is characteristic of all plethodontid salamanders). This "walk" requires both the male and the female to modulate their courtship behaviors in response to the other partner (Arnold 1976
). After the pair has been in the tail-straddling walk, the pair halts and the male lowers his cloaca to the substrate and begins to deposit a spermatophore. Deposition takes approximately 5–7 min, and if sperm transfer is to occur, the female must be sufficiently receptive to remain in contact with the male during that time. Thus, it is critical for the male to use the female's behavior to judge the level of her receptivity and then time his spermatophore deposition accordingly. If a female does not appear to be sufficiently receptive, the male has the option of delivering pheromones to the female during the courtship. These courtship pheromones increase the female's receptivity (judged by courtships that lead to spermatophore deposition more quickly), as compared with courtships in which the female only received a saline control (Rollmann et al. 1999, 2003). Thus, the duration of courtship (from the female entering into tail-straddling walk to spermatophore deposition) is an indirect measure of female receptivity, and receptivity can be influenced by male courtship pheromones.
Plethodontid male courtship pheromones are secreted from specialized mental (submandibular) glands that hypertrophy seasonally (Sever 1976). In our 2 main study species (Plethodon shermani and Desmognathus ocoee), we have extracted the pheromones expressed in the male mental glands and experimentally delivered these protein pheromones to female salamanders. In each of these species, experimental delivery of conspecific courtship pheromones decreased courtship time (Houck and Reagan 1990; Rollmann et al. 1999; Houck et al. 2007). Pheromone delivery and specialized mental glands occur in the majority of the >380 species within the family Plethodontidae. In fact, courtship pheromone delivery in plethodontids apparently has occurred for over 100 million years (see Houck and Arnold 2003).
In P. shermani, the delivery of courtship pheromones typically occurs during tail-straddling walk. The male temporarily halts and turns back toward the female (while the female continues to straddle his tail) and "slaps" his gland on her nares (Arnold 1976). This slapping behavior typically is repeated many times during tail-straddling walk. The pheromones enter the female's nasal cavity (Dawley and Bass 1989) and stimulate cells in the vomeronasal organ (Wirsig-Wiechmann et al. 2002, 2006). Biochemical characterization of the gland extract of male P. shermani revealed that a 22-kDa protein, termed plethodontid receptivity factor (PRF), was highly expressed in male mental glands (Rollmann et al. 1999; Fontana et al. 2007). A purified solution containing multiple PRF isoforms increased female receptivity when experimentally delivered to her nares (Rollmann et al. 1999). Multiple isoforms of PRF are expressed in an individual male's gland, and PRF shows significant amino acid variation at multiple levels: individual, population, and species (Rollmann et al. 2000; Watts et al. 2004; Palmer et al. 2005).
We hypothesized that the recombinant expression of PRF would verify that a single isoform can influence female receptivity. In this study, we report 1) the development of an expression system to produce a functionally active recombinant pheromone protein and 2) the results of a behavioral experiment that tested whether the expressed protein alone could modify female behavior.
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Materials and methods |
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Recombinant protein preparation
Recombinant PRF (termed rPRF) was expressed as a precursor protein with a cleavable N-terminal 6xHis affinity tag. We chose the P. shermani PRF isoform 3 (GenBank accession number AF181482) for expression because this isoform is abundantly expressed in the mental glands of males in our study population from Macon County, North Carolina. Isoform 3 was amplified by polymerase chain reaction with primers designed to attach a KpnI restriction site and the sequence for an enterokinase cleavage site (DDDDK) immediately before the initial Glu1 residue. Downstream, we attached a stop codon immediately after the final codon and then a HindIII restriction site. This construct was inserted into the KpnI/HindIII sites of pET32(a) (Novagen, San Diego, CA), and then this expression plasmid was transformed into an Escherichia coli strain CodonPlus-(DE3)-RIPL (Stratagene, La Jolla, CA) for protein expression.
Bacterial expression of soluble rPRF was performed at 25 °C with high efficiency (
100 mg/l). Cultures (Terrific Broth supplemented with 100 µg/l ampicillin) were grown using standard methods and with overnight protein expression induced by 0.1 mM isopropyl-β-D-thiogalactopyranoside. The bacteria were harvested by centrifuging and stored at –80 °C.
Initial purification of the soluble rPRF fusion protein was conducted using His-Bind Resin (Novagen) at 4 °C and with buffers and methods described in the supplied protocol. Briefly, the bacteria were resuspended in binding buffer (5 mM imidazole) supplemented with DNase (10 µg/ml) and lysozyme (10 mg/ml) and then lysed in an Emulsiflex/C5 High Pressure Homogenizer (Avestin, Inc., Ottawa, Canada). The lysate was sonicated and clarified by centrifuging, and the 20–90% ammonium sulfate saturation fraction was then obtained using standard procedures. The protein was resuspended in binding buffer and applied to the His-Bind Resin (50 ml bed volume). The column was washed with 6 successive column volumes of binding buffer, 6 of wash buffer (60 mM imidazole), and 3 of elution buffer (1 M imidazole). Final purity was estimated at >95%, and the yield was measured at over 100 mg/l of bacterial culture.
Recombinant enterokinase (EMD Biosciences, Madison, WI) was used to cleave the His-tag. Cleavage products were separated from rPRF by HLPC (99% purity). After cleavage, the final rPRF protein was shown by mass spectral analyses to have a mass (21,641 Da) identical to the native isoform (isoform 3 in Rollmann et al. 1999). The protein concentration of the rPRF solution was adjusted to the final concentration of 0.7 µg/µl in 0.5x phosphate-buffered saline (PBS) using ultra filtration.
To characterize the secondary structures of rPRF and the native PRF isoform 3, circular dichroism (CD) analyses were performed using a 0.1-cm path length cell at 0.2-nm intervals with 5 scans (182–260 nm) averaged at 20 °C (Jasco J-810 Spectropolarimeter). Far-UV CD spectra of high-performance liquid chromatography–purified rPRF and PRF isoform 3, in 20 mM potassium phosphate buffer (pH 7.4), were taken at a protein concentration of 0.11 and 0.04 µg/µl, respectively. The resultant spectra were corrected for the buffer signal and interpreted with Olis GlobalWorks algorithms (www.olisweb.com).
Courtship trials
Male and female P. shermani in breeding condition were collected from Macon County, North Carolina (035°10'48''N, 083°33'38''W) during August 2005. Animals were collected with the appropriate permits from North Carolina Department of Wildlife. The animals were housed at Oregon State University for the duration of the behavioral observations, which were staged over 8 nights between 7 and 19 September 2005. Methods and animal care were approved by Oregon State University's Institutional Animal Care and Use Committee (LAR 3007 to L.D.H.). Animal care followed Houck et al. (1998). Briefly, animals were individually housed and kept on a natural photoperiod. Each animal was fed waxworms (Galleria mellonella) weekly.
Before courtship trials were staged, animals were randomly assigned to pairs and prescreened (to determine their propensity to court in a laboratory environment) by allowing each pair to remain together in the same box overnight. The following morning, the animals were put back in their maintenance boxes. Each courtship box was checked for the presence of a spermatophore base, which was taken as an indication of a successful courtship. Most animals were given 3 courtship encounters, and animals that mated at least once were selected to be used in the courtship trials. This selection process favored females that had a higher likelihood of mating in the laboratory.
To ensure that females only received pheromones delivered by the researchers, we anesthetized each male used in the courtship trials and surgically ablated its mental gland. Deglanded males included in the experiment were given at least 2 weeks to recover before they were used in behavioral trials. Males fully recover and court normally after this procedure (Houck LD, unpublished data).
Once reproductively active animals were selected, they were randomly reassigned to different male–female pairs before the onset of the courtship trials. During a trial, each male–female pair was placed in a clear plastic box (9 x 17 x 30 cm) under low light conditions. Trials were conducted during the time of night when the animals normally would be found courting in the field (2200 to 0100 h).
The delivery of either rPRF or a control saline solution to each female followed the protocol of Houck et al. (2007). To summarize, the onset of experimental pheromone delivery was designed to coincide with a male's natural pheromone delivery behavior to control for tactile stimulation. Once a pair was engaged in tail-straddling walk and the deglanded male had attempted pheromone delivery by touching the female's nares, either rPRF (0.7 µg/µl in 0.5x PBS, a concentration used in prior behavioral experiments) or 0.5x PBS was delivered to the female using a micropipette. On a given observation night (=trial), the first type of treatment (rPRF or 0.5 x PBS) was randomly assigned to the first female to engage in tail-straddling walk. Subsequent treatments alternated between rPRF and 0.5x PBS such that both treatments were given to multiple females, but an individual female received only one type of treatment. Overall, each treated female received a total of 15 µl of solution onto her nares (5 µl every 10 min). Due to temporal limitations (each observer can only keep track of a limited number of courting pairs at the same time, and trials need to be staged during the peak courtship season), we were unable to test multiple stimuli (e.g., rPRF and native PRF) during the same courtship season.
During each trial night, observers recorded observations using focal animal sampling (Altmann 1974). As a measurement of courtship duration, we recorded the time from when a female entered tail-straddling walk to the termination of spermatophore deposition because these behaviors were unambiguous and easily scored by all observers. For all pairs (pheromone and saline treated), we also recorded the number of slaps that a male administered during courtship and whether the female was inseminated.
Data analysis
Data on the mean duration of courtship were analyzed using a 1-tailed t-test with alpha = 0.05. We used a 1-tailed test based on results from prior behavioral experiments showing that PRF pheromones significantly increased female receptivity and thus resulted in a reduced courtship duration (e.g., Rollmann et al. 1999). Data on the average number of slaps (males attempted pheromone delivery even though they were deglanded) were analyzed using a 2-tailed t-test with alpha = 0.05. Data on insemination success for pheromone-treated and saline-treated females were analyzed using a 2 x 2 contingency table with alpha = 0.05.
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Results |
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The bacterial expression system and subsequent purification produced a single isoform of rPRF. Due to sequence similarity with the interleukin (IL)-6 family of cytokines (4
-helix bundle structures; Rollmann et al. 1999), rPRF was expected to have a high -helical content similar to that of the native isoforms. The CD spectrum shown in Figure 1 indicated a predominately -helical structure, particularly the trough between 208 and 222 nm, which is similar to the documented structure of known secondary structures of the IL-6 cytokines (e.g., Somers et al. 1997). The helical contents of the native PRF (isoform 3) and rPRF were identical (Figure 2) within typical experimental conditions (Table 2).
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rPRF decreased the mean courtship time by approximately 14 min (21%): mean of saline treatment = 66 min and mean of rPRF treatment = 52 min. Thus, the behavioral assay confirmed that rPRF increased female receptivity (t26 = 3.04, P
0.01, 1-tailed t-test) in P. shermani (Figure 2).
The only effect of rPRF on a specific behavior pattern was the duration of tail-straddling walk (i.e., time from commencement of tail-straddling walk to spermatophore deposition). There was no difference between the treatment and the control groups in 1) the mean number of times males attempted to deliver pheromones during tail-straddling walk (t26 = 0.112, P < 0.911, 2-tailed t-test) or b) the relative number of females in each treatment group that were inseminated (
21 = 0.022, P > 0.882).
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Discussion |
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In P. shermani salamanders, the average courtship duration of male–female pairs was significantly reduced by the experimental delivery of a single recombinant isoform of the male courtship pheromone PRF. This behavioral response to pheromone delivery represents one of the first demonstrations in vertebrates that a nonpeptide protein can be synthesized and still retain the full effects of the native pheromone protein (for a major urinary protein [MUP] example, see Chamero et al. 2007
).
The behavioral effect of rPRF (21% reduction in average courtship duration) is similar to effects documented in 2 behavioral tests previously conducted with P. shermani courtship pheromones. In comparison with the current results, rPRF mimicked the activity of 1) a purified blend of multiple native PRF isoforms: 15% reduction in courtship time (Rollmann et al. 1999) and 2) the combined proteins contained in pooled secretions obtained from multiple male mental glands: 22% reduction in courtship time (Rollmann et al. 2003). The difference in average courtship durations among these 3 experiments (21%, 15%, and 22%) most likely is due to experimental variation in different years (e.g., minor differences in the relative timing of prescreening animals for courtship propensity and of staging courtship trials). In short, the results of behavioral tests over multiple years confirm that PRF consistently had significant effects on female receptivity.
We interpret the effects of PRF primarily in terms of sexual selection, as the courtship pheromone functions to influence the female's mate choice. In fact, the male's repeated delivery of PRF apparently acts physiologically to focus the female's attention, in effect "persuading" her to continue mating with him. When the male judges that the female is sufficiently receptive (presumably from some aspects of the female's behavior; Arnold 1977), only then will he begin the process of spermatophore deposition. Thus, as this chemical persuasion is successful, the male is more likely to inseminate the female. The significance of pheromone delivery is realized when one observes the actions of a female that already is receptive (prior to her interactions with a male). In this situation, the female actually will approach the male and immediately initiate the tail-straddling walk. In these rare cases, the male completely eliminates courtship pheromone delivery and moves rapidly into spermatophore deposition (Houck, personal observations; and see Halliday 1975). Thus, a pheromone that increases the female's interest in the male can be critical to the male's mating success. In addition, a mating pair that spends less time in tail-straddling walk is more likely to avoid disruption of the courtship (e.g., by rival males) or (reflecting natural selection here) more likely to avoid becoming the target of local predators (Houck 1986).
These effects of PRF on mate choice and mating success are important but still do not reflect the full story of the PRF signal and the female response to this signal. More light is shed on the nature of the PRF pheromone by comparing changes in the amino acid sequences of PRFs not only within and between populations but also across a multispecies Plethodon lineage that spans at least 15 million years of divergence. These molecular comparisons reveal that PRF has changed substantially (in terms of amino acid sequences) from species to species, resulting in highly significant variation in the male signal (Rollmann et al. 2000; Watts et al. 2004; Palmer et al. 2005). This kind of extraordinary variation also has been documented in certain other vertebrate chemical signals (Emes et al. 2004). High numbers of pheromone polymorphisms, for example, are found in the MUPs of mice and other rodents: over 2000 different MUP sequences have been deposited in GenBank (Beynon and Hurst 2004). The complex mixture of MUPs apparently confers information about individual identity (Hurst et al. 2001), rather than eliciting a specific behavioral response (but see Novotny, Ma, et al. 1999; Chamero et al. 2007). Another highly variable pheromone signal has been described in male mice: a 7-kDa peptide (transcribed from a multigene family that contains at least 23 genes) is secreted from the extraorbital lacrimal gland to convey information to females (Kimoto et al. 2005). These examples have in common the likelihood that sexual selection is acting on the male signal through the powerful agent of female mate choice (for further details, see Watts et al. 2004). In contrast, variation in the courtship pheromone signal of certain male newts more likely is a result of natural selection, not sexual selection. In these Cynops species (see Table 1), variation has been documented for the decapeptide pheromone sodefrin (Nakada et al. 2007). Males in 2 separate populations express different variants of this decapeptide, and females are more stimulated by the pheromone produced by males from their own population.
Whether or not these pheromone signals are highly variable, peptides and proteins appear to be important nonvolatile chemical signals, particularly in amphibians—the first vertebrate group to possess a discrete vomeronasal organ. Kikuyama et al. (2002) noted that amphibian pheromones are predominantly amino acid based, and this trend is supported by the recent research (Palmer et al. 2005; Houck et al. 2007). In studying these peptides and proteins, synthesis presents unique challenges: expression in many cell lines is not difficult but signal effectiveness also is determined by the correct posttranslational modifications and 3-dimensional conformation. Some proteins that act as pheromones in the native form have lost partial or full activity when recombinantly expressed. Purified MUPs had a puberty-accelerating effect on female mice, but recombinant MUPs did not (Novotny, Ma, et al. 1999). In hamsters, a cloned version of the aphrodisin pheromone did not elicit mating behavior from males (Jang et al. 2001), although the native molecule had behavioral effects (Singer et al. 1986). Also, Singer et al. (1976) claimed that male hamsters still showed some response to synthesized dimethyl disulfide, but reactions were only 20–40% of the response to the native vaginal secretions and the absence of any smaller, bound molecules was not demonstrated. Together, these results suggest that context dependence is important, and thus, the increasing complexity of social behaviors in some mammalian groups may have necessitated more complex cues to aid social communication (for review, see Johnston 2003).
The expression system we have developed for PRF isoforms will allow us to test the effectiveness of individual isoforms, as well as isoform combinations. We also will be able to test whether females have different isoform preferences, as suggested by the extreme isoform variation already documented for P. shermani. In addition, this system can help determine which sites in the protein are important for biological activity, using techniques such as site-directed mutagenesis. Finally, manipulating PRF isoforms may help us gain an understanding of the evolutionary processes that produce and maintain protein polymorphisms.
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Funding |
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National Science Foundation Grants (IOB-0110666, IOB-0416724 to L.D.H., IOB 0416834 to R.C.F.).
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Acknowledgements |
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We thank Elyse Vaccaro and Stacey Smith for assistance in collecting animals and with behavioral observations and Audrey Thompson for assistance with protein purification. We also thank the Directors and Foundation of the Highlands Biological Station for providing facilities and support for our fieldwork and research.
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Accepted 24 April 2008
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