Abstract
Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10−12 mol l−1 for 3-keto petromyzonol sulfate and 10−10 mol l−1 for the other bile acids. At concentrations above 10−9 mol l−1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Bile acids are potent stimulants for both the gustatory and olfactory systems of many fish species (Hara 1994; Li et al. 1995; Michel and Lubomudrov 1995). Initially, it was proposed that the bile acids are appealing candidates for salmon pheromones due to their structural diversity and stability (Døving et al. 1980; Stabell 1987). Furthermore, electrophysiological studies demonstrated that an odotopic map of responses to bile acids and amino acids are represented in different regions of olfactory bulbs of chars (Salvelinus alpinus) and graylings (Thymallus thymallys) (Døving et al. 1980). Behavioral studies in Atlantic salmon (Salmo salar) also showed that intestinal extracts, which are known to contain bile acids, induced strain-specific preference and searching behaviors (Stabell 1987). Indeed, several experiments indicated that a number of bile acids induced behavioral responses in fish (Jones and Hara 1985; Hellstrøm and Døving 1986; Sola and Tosi 1993; Hara 1994). More recently, it has been shown that bile acid profiles of lake trout (Salvelinus namaycush) are largely influenced by sex and maturation stage (Zhang et al. 2001) and that bile of female rainbow trout (Oncorhynchus mykiss) contains a pheromone (Vermeirssen and Scott 2001). However, no specific bile acids have been identified as pheromones in teleost fish.
Recently, the sea lamprey (Petromyzon marinus) has emerged as an efficient animal model for studies of bile acids as possible pheromones because it is the only species in which specific bile acids have been linked to specific pheromone functions. Biochemical and behavioral studies have shown that sea lampreys at different life stages release specific bile acids that induce characteristic behaviors in adults (Bjerselius et al. 2000; Li et al. 2002). This species experiences three stages during its life cycle. The sea lamprey inhabits tributary streams during the larval stage, enters the Atlantic Ocean or large lakes (e.g., the Laurentian Great Lakes) to feed after metamorphosis into the parasitic stage and returns to streams in the adult stage to spawn and die (Hardisty and Potter 1971). Larval sea lampreys release two bile acids, petromyzonol sulfate (PZS) and allocholic acid (ACA) (Haslewood and Tokes 1969; Li et al. 1995; Porkinghorne et al. 2001; Fig. 1A and B), that are potent stimulants to olfactory organs (Li et al. 1995; Li and Sorensen 1997) and induce locomotion behaviors in adults of the same species in a laboratory maze (Bjerselius et al. 2000; Vrieze and Sorensen 2001). Furthermore, adult males, after onset of spermiation, release two bile acids, 3-keto petromyzonol sulfate (3kPZS; Li et al. 2002; Fig. 1C) and 3-keto allocholic acid (3kACA; Yun et al. 2003; Fig. 1D). 3kPZS has been shown to induce increased swimming activity in ovulating females and their ultimate attraction (Li et al. 2002). The function of 3kACA has not been determined and is suspected to be a minor component of the male pheromone (Yun et al. 2003).
These four bile acids present a physiological challenge for the olfactory system of adult sea lampreys that use them as possible pheromones. Sea lamprey adults spawn in rapids where current velocity typically reaches 1 m s−1 (Applegate 1950). Bile acids released by males would be rapidly diluted. Moreover, the adult male bile acids, 3kPZS and 3kACA, only differ from their larval counterparts (PZS and ACA) by having a carbonyl, as opposed to a hydroxyl at carbon 3 (Fig. 1). Yet larvae inhabit spawning streams year round (Moore and Schleen 1980) and release PZS and ACA when spermiating males release 3kPZS and 3kACA to attract ovulated females. It is essential for mature female sea lampreys to be able to distinguish male bile acids from larval ones. Further, it has yet to be shown directly that 3kPZS and 3kACA stimulate the olfactory epithelium of adult females.
In this study, our objectives were to determine electrophysiologically (1) the potency of 3kPZS and 3kACA to the olfactory organs of adult females, and (2) if the olfactory epithelium of females discriminates among the four bile acids. Our electrophysiological data from this study indicate that 3kPZS, demonstrated to function as a male pheromone (Li et al. 2002), represents a highly potent and distinct odor to adult female sea lampreys.
Materials and methods
Experimental fish
Adult sea lampreys were collected from tributaries to lakes Huron and Michigan between April and July 2000–2002 by the staff of the US Fish and Wildlife Service, Marquette Biological Station, Marquette, Michigan, USA. Lampreys were transported to the main laboratory at the US Geological Survey, Hammond Bay Biological Station, Millersburg, Michigan USA. Females were separated from males and held in flow-through tanks (1000 l) with chilled Lake Huron water at temperatures ranging from 6°C to 8°C. Chilled water slows senescence, preserving measurable olfactory responsiveness throughout the spawning season.
Test stimuli
The olfactory epithelia of female sea lampreys were exposed to solutions of l-arginine (Sigma Chemicals, St. Louis, Missouri, USA), four bile acids (ACA, PZS, 3kACA, 3kPZS; Toronto Research Chemicals, ON, Canada; all >97% pure), and water conditioned with spermiating male washings (SMW). Stock solutions of bile acids were made at concentrations of 10−3 mol l−1 using de-ionized water or methanol and stored at –20°C. A 10−2 mol l−1 l-arginine standard stock solution was made with de-ionized water every week and stored at 4°C.
To collect SMW, males were held in flow-through tanks (200 l; 15 males per tank) with heated Lake Huron water at temperatures ranging from 15°C to 18°C. These temperatures were used to promote spermiation. Males were checked for spermiation according to the criteria set forth by Siefkes et al. (2003a). Water was conditioned with spermiating male sea lampreys by holding individual males for 4 h in polyethylene buckets filled with 10 l of Lake Huron water. These buckets were aerated and kept in a water bath of 18°C. The SMW were either used immediately in or stored at −80°C for electro-olfactographic analyses. The SMW were measured for 3kPZS concentration using an enzyme-linked immunosorbent assay (ELISA; Yun et al. 2002), but were not measured for the other bile acids used in this study or for any other compounds.
Electro-olfactogram recording procedure
The same source of Lake Huron water was used to maintain the animal, collect male lamprey odor, dilute bile acids, and perfuse the naris during recording.
Female sea lampreys were tested for olfactory sensitivity to the four bile acids and SMW described above. Electro-olfactogram (EOG) recording was performed as described by Li et al. (1995). Briefly, sea lampreys were anesthetized with an intramuscular injection of metomidate hydrochloride (3 mg kg−1 body weight; Syndel, Vancouver, BC, Canada), immobilized with an intramuscular injection of gallamine triethiodide (150 mg kg−1 body weight; Sigma, St. Louis, Missouri, USA) and placed in a water-filled trough. The head of the female remained above the water and the gills were supplied with aerated water. The olfactory lamellae were then exposed and perfused with water. Differential electrical potential between the skin surface and the sensory epithelia in response to each test stimulus were recorded using two Ag/AgCl electrodes (type EH-1S, World Precision Instruments, Sarasota, Florida, USA) filled with 3 mol l−1 potassium chloride and bridged with 8% gelatin:0.9% saline-filled glass capillary tubes. The recording electrode was placed between two lamellae and was adjusted to maximize the response to the L-arginine standard while minimizing the response to a blank water control when the reference electrode was placed on the skin near the naris. Electrical signals were amplified and digitized by a Power Lab (ADI Instruments, Castle Hill, NSW 2154, Australia) and displayed on a computer.
Concentration-response relationships
Stock solutions of odors were diluted in Lake Huron water immediately before testing. To determine concentration-response relationships, a 10−5 mol l−1 l-arginine standard was introduced into the olfactory epithelium of a female for 5 s and the EOG response measured to establish a reference of electrical activity (Li et al. 1995). Next, blank water control was introduced and the response measured to confirm the absence of response from the water supply. Increasing concentrations of bile acids starting at 10−13 mol l−1 and SMW were then introduced and the responses measured. Measuring the response to the l-arginine standard and blank water control again at the end of the dilution series concluded each trial. The epithelium of each female was allowed to recover for at least 3 min between stimuli, and each concentration of the test stimuli was assayed at least twice. EOG response magnitudes from females were measured in millivolts and expressed as a percentage of the response to the l-arginine standard.
Cross-adaptation
Cross-adaptation, developed by Caprio and Byrd (1984), was used to compare the EOG response to a test stimulus before and during adaptation to an adapting compound using a protocol by Li and Sorensen (1997). In a given trial, baseline olfactory EOG responses of females to blank water control (control A), an l-arginine standard and test stimuli (SMW and all four bile acids; initial response) were recorded. The test stimuli were used at concentrations that elicited approximately equipotent olfactory responses at about 100% of the l-arginine standard. During adaptation, the olfactory epithelium of a female was continually exposed to the adapting stimulus for 5 min after which a 5-s application of the same adapting stimulus was tested, first at the concentration used in the adaptation (control B) and then at twice the concentration used for the adaptation (self-adapted control). Then the other test stimuli were tested in the adapting solution (adapted response). Each test was interspersed with 5-s applications of the l-arginine standard and control B to confirm the responsiveness of the female. Switching the adapting stimulus back to blank water completed the trial. The epithelium of the female was allowed to recover for 30 min and then the female was tested again using another adapting stimulus. Cross-adaptation data were expressed as percent initial response (PIR) using the following formula adapted from Caprio and Byrd (1984) and Li and Sorensen (1997):
where a larger PIR indicates less cross-reactivity between olfactory receptor mechanisms or separate receptor sites and a low PIR indicates more cross reactivity or shared receptor sites and/or a common signal transduction pathway.
Statistical analysis
Statistical analysis system (SAS) software was used to conduct all analyses. In concentration-response experiments, responses were visually compared. The lowest concentration at which a stimulus elicited a response larger than the blank water control (Student’s t-test) was considered to be its response threshold.
In cross-adaptation experiments, a paired t-test was used to determine if responses to a test stimulus during adaptation were significantly different than the responses to the same test stimulus before adaptation. All PIR data were subsequently analyzed to directly detect cross-reactivity by subjecting all PIR data to a two-way analysis of variance (ANOVA). If the main effect was found to be significant, the PIR data was divided into five groups according to adapting stimulus. For each group, responses of adapted epithelia to test stimuli were analyzed by a one-way ANOVA to determine the affect of the adapting stimulus. Again, if the main effect was significant, the significance of the adapting effect for each test stimulus was determined by comparing all PIRs in the group with the PIR of the self-adapted control using Dunnett’s test, which tests for differences between several treatments and a single control.
The following categories were used to classify cross-adaptation responses: (1) not adapted, meaning responses during adaptation were not significantly different from the initial response (paired t-test, P>0.05); (2) partially adapted, meaning responses during adaptation were significantly less than the initial responses (paired t-test, P<0.05), but the PIR were significantly greater than the control (Dunnett’s, P<0.05); and (3) adapted to control levels, meaning PIR were not significantly different than the control (Dunnett’s, P>0.05).
Results
All chemicals tested were stimulatory to the olfactory epithelium of female sea lampreys. The mean response to the l-arginine standard was 0.859±0.04 mV (mean±SE). Concentration-response relationships of the four bile acids were plotted together as percentages of the l-arginine standard (Fig. 2A). Responses to all concentrations of bile acids ranged from –9% to 640%, and the blank water control elicited a mean response of 7.9±6.5% (mean±SE). 3kPZS and PZS had similar concentration-response curves that were exponential in shape with steep slopes between 10−10 and10−6 mol l−1. The response threshold, or the lowest concentration that elicited a response significantly greater than the blank water control, for 3kPZS was 10−12 mol l−1 (31.4±3.2%, mean±SE; Student’s t-test, P<0.01; Fig. 2C) and for PZS was 10−10 mol l−1 (26.8±9.6%, mean±SE; Student’s t-test, P<0.01; Fig. 2C). 3kACA and ACA had similar concentration-response curves that were linear in shape from the response threshold of 10−10 mol l−1 (37.8±20.5%, mean±SE; Student’s t-test, P<0.01 and 30.0±7.5%, mean±SE; Student’s t-test, P<0.01, respectively; Fig. 2C) to 10−6 mol l−1, the highest concentration tested. The concentration-response curves for 3kACA and ACA were shallower than the curves generated with 3kPZS and PZS.
When comparing the concentration-response curves of SMW and 3kPZS, the curve for SMW was plotted according to the concentration of 3kPZS measured in the SMW samples using ELISA (Yun et al. 2002). The concentration-response relationship of SMW had an exponential shape similar to the 3kPZS curve (Fig. 2B). Also, both SMW and 3kPZS had response thresholds of 10−12 mol l−1 (16.2±3.2%, mean±SE; Student’s t-test, P=0.05 and 31.4±3.2%, mean±SE; Student’s t-test, P<0.01, respectively; Fig. 2D). Although the curves were similarly shaped, the SMW curve generated slightly larger responses at equivalent 3kPZS concentrations above the response threshold.
The responses to the l-arginine standard did not change before and during adaptation to bile acids (Fig. 3; paired t-test, P>0.10), but those to bile acids did (two-way ANOVA, P<0.01). When used as the adapting stimuli, three of the four bile acids completely adapted themselves (3kPZS, PZS and ACA; Fig. 3; paired t-test, P<0.01, initial responses compared to responses during adaptation; also visually compared). Although, the response to 3kACA was significantly lower than the initial response (paired t-test, P<0.01), 3kACA appeared to not completely adapt itself (Fig. 3B). The analyses revealed three patterns of cross-reactivity among the four bile acids. First, when 3kPZS was used as an adapting stimulus, the response to PZS was only partially adapted, and vice versa (Dunnett’s, P<0.05; paired t-test, P<0.01; Fig. 3A and C). Second, the responses to 3kACA and ACA were not adapted to 3kPZS (paired t-test, P>0.10; Fig. 3A) and only partially adapted to PZS control levels (Dunnett’s, P<0.05; paired t-test, P<0.03; Fig. 3C). Similarly, responses to 3kPZS and PZS were not adapted to either 3kACA or ACA control levels (paired t-test, P>0.05; Fig. 3B and D). Third, when ACA was used as an adapting stimulus, the response to 3kACA was adapted to control levels, and vice versa (Dunnett’s, P>0.05; Fig. 3B and D). When SMW was used, all bile acid stimuli were adapted to control levels (Dunnett’s, P>0.05; Fig. 3E).
Discussion
This study establishes that the olfactory organs of adult female sea lampreys are highly sensitive to and can electrophysiologically discriminate bile acids released by males and larvae of the same species. Concentration-response curves showed that both male and larval bile acids were highly stimulatory to female olfactory organs and that there was a wide range of response dynamics associated with the four tested bile acids. In the lower range of concentrations tested, the main component of the male pheromone (Li et al. 2002; Yun et al. 2002), 3kPZS, with a response threshold of 10−12 mol l−1, was 100 times more potent than the other three bile acids, whose response thresholds were 10−10 mol l−1. This difference in potency may be advantageous and critical for 3kPZS to function as a male pheromone for two apparent reasons. First, during the spawning season, adults and larvae occupy the same streams (Moore and Schleen 1980) in which PZS and ACA are present at concentrations between 10−11 and 10−12 mol l−1 (Porkinghorne et al. 2001), which are below the detectable concentrations by female adults. Unless the real response thresholds are lower than those determined by our EOG recording, PZS and ACA should not interfere with female detection of 3kPZS in spawning streams. Second, sea lampreys spawn in fast flowing rivers and streams where current velocity typically reaches 1 m s−1 (Applegate 1950). Bile acids released by males would be quickly diluted by the large volume of water passing by the male. Even though 3kPZS is produced and released at a very high rate (ca. 0.50 mg per male per hour; Yun et al. 2002, 2003) through a highly efficient and specialized mechanism (Siefkes et al. 2003b), a low response threshold for 3kPZS would be advantageous for its pheromone function.
It is notable that in a previous study (Li et al. 1995), the olfactory epithelia of sea lamprey migratory adults of both sexes showed EOG responses to PZS and ACA at concentrations as low as 10−11 and 10−12 mol l−1, which were lower than the 10−10 mol l−1 determined in this study (Fig. 2A), and which were similar to the concentrations measured in spawning streams (Porkinghorne et al. 2001). The difference in measured response thresholds is probably due to the different animal and experimental conditions between the two studies. The animals used in Li et al. (1995) were migratory adults captured very early in their spawning migration whereas animals used in our study were often captured later in the migration. It has been shown that the EOG responsiveness of adult sea lampreys to larval bile acids gradually decreases as the spawning migration progresses (Li 1994). Furthermore, the location, water, holding tanks, odor delivery system, digitizer, and amplifier used in the study by Li et al. (1995) were different from those used in this study. Nonetheless, these two studies indicate that sea lampreys are highly sensitive to bile acids released by individuals of the same species.
Our cross-adaptation experiments demonstrated that the olfactory epithelium of female sea lampreys electrophysiologically discriminate between different bile acids produced by males and larvae of the same species. It appears that, from the patterns of cross adaptation, 3kPZS and PZS represent different odors to the females. In contrast, 3kACA and ACA appear to represent the same odor or two very similar odors. Further, the odor of 3kACA and ACA is very different from those of 3kPZS or PZS. In other words, 3kPZS represents a unique odor among bile acids tested in this study, which may very well be another advantageous physiological feature to promote the functionality of 3kPZS as a pheromone. The distribution of lamprey larvae in streams is not random; rather, it is clumped (Applegate 1950). Thus, it is possible that at certain locations the concentrations of PZS and ACA are higher than the average. Ovulating females, in search of spermiating males through the 3kPZS signal (Li et al. 2002, Siefkes et al. 2003a), may be lost in locations with concentrated larvae without the ability to discriminate 3kPZS from other conpecific bile acids.
Another issue is that females also need to discriminate bile acids released by other aquatic and terrestrial animals. Li and Sorensen (1997) showed that PZS is electrophysiologically discriminated by adult lamprey from all other common animal bile acids tested. Since 3kPZS shares with PZS a combination of two molecular features (5α-configuration and a sulfate ester at carbon 24; Haslewood and Tokes 1969; Li et al. 2002), which has been found only in lamprey bile acids, it is likely that 3kPZS is also readily discriminated from bile acids of other animals. However, the direct evidence supporting this speculation can only come from direct testing using 3kPZS.
There were two curious phenomena that we observed during our cross-adaptation experiments. One was the apparent lack of self-adaptation when 3kACA was used as the adapting stimulus. Contamination of the 3kACA controls may explain the lack of self-adaptation, but this is unlikely as can be seen by the tight error bars around the self-adapted control (Fig. 3B), the complete adaptation to ACA (Fig. 3D), and the purity of the compound. Explanation may prove to be difficult. The other phenomenon is the evidence of non-reciprocal adaptation when 3kPZS and PZS are used as the adapting stimulus. It appears that PZS as an adapting stimulus partially adapts 3kPZS by around 60%, while 3kPZS partially adapts PZS only by around 40%. Whether this pattern of non-reciprocal adaptation is indicative of more receptor sites for PZS than 3kPZS has yet to be determined. The lack of self-adaptation of 3kACA and the non-reciprocal adaptation of 3kPZS and PZS need to be explored further. Nonetheless, the other cross adaptation data indicate that 3kACA/ACA, 3kPZS and PZS represent different odors to females, and support the previous hypothesis that 3kPZS functions as a pheromone (Li et al. 2002).
Whether 3kACA could function as a component of the male pheromone remains elusive. This compound is a potent stimulant for females (Fig. 2A) and is released only by the male lamprey during spermiation (Yun et al. 2003), exactly the same condition when 3kPZS is released (Li et al. 2002). However, 3kACA is completely adapted to control levels by ACA, and vice versa. It is likely a behavioral study could conclude on its potential function as a pheromone component.
EOG data from this study also implicate that a change at carbon 3 may largely influence the odor quality of bile acids. Several previous studies have clearly indicated that conjugating groups (or the lack thereof) are critical in the lack of cross-adaptation of bile acids by fish olfactory epithelia. In zebrafish (Danio rerio), taurine-conjugated bile acids are more stimulatory than free or glycine-conjugated bile acids (Michel and Lubomudrov 1995). Lake char (Salvelinus namaycush) appear to have several specific olfactory receptor subtypes that distinguish between free, taurine-conjugated and carbon-3 sulfated bile acids (Zhang et al. 2001; Zhang and Hara 1994). Similarly, migratory adult sea lampreys appear to electrophysiologically discriminate free, carbon-24 taurine-conjugated, carbon-3-sulfated and carbon-24-sulfated bile acids (Li and Sorensen 1997). All these results are corroborated by our discovery in this study that two free bile acids (ACA and 3kACA) are not completely cross-adapted by the two carbon-24-sulfated bile acids (PZS and 3kPZS). In addition, it is noteworthy that our data clearly indicate that a carbon-3 carbonyl or hydroxyl, neither of which is a conjugating group, also are critical to the female lamprey olfactory epithelium in electrophysiological discrimination of conspecific bile acids.
Our experiments using SMW have yielded mixed results. SMW is known to induce characteristic behavioral responses in ovulating females (Li et al. 2002) and to contain 3kPZS and 3kACA at a ratio of 25 to 1 (Li et al. 2002; Siefkes et al. 2003b; Yun et al. 2002, 2003). Corroborating with this discovery, concentration-response curves of SMW and 3kPZS were similar in shape and magnitude (Fig. 2B), especially in the lower range of concentrations. It is likely that, at lower concentrations, the SMW potency is largely attributable to 3kPZS. The slightly larger response magnitudes at concentrations above 10−10 mol l−1 is probably largely due to 3kACA. Presumably, SMW also contains many other compounds that might be odorous and may contain the other bile acids used in this study (Fig. 3E). Previous chemical and electrophysiological studies indicated that other compounds are likely to only be present in and not likely to be stimulatory at low concentrations (Li et al. 2002; Yun et al. 2002, 2003). Further, when collected under a condition similar to that used in this study, water from spermiating males is stimulatory when diluted 106 times whereas water from pre-spermiating males is stimulatory only when diluted 102 times or less (Li 1994), and the main difference between the two waters is the presence of 3kPZS and 3kACA from spermiating males (Li et al. 2002; Yun et al. 2002).
Our cross adaptation experiments using SMW, however, do not clearly support the notion that 3kPZS is the only stimulatory compound in diluted SMW. When SMW was used as an adapting stimulus, the response to 3kPZS was adapted to control levels, which was expected. In addition, SMW also adapted PZS and ACA to control levels, which was not expected. SMW may contain trace amounts of PZS and ACA (if any, and only at an amount that is less than 1% of that of 3kPZS; Yun et al. 2002), which is not likely to be sufficient to suppress responses to PZS and ACA at the concentrations used. This issue needs to be clarified in future experiments.
In conclusion, the olfactory epithelium of adult female sea lampreys is highly sensitive to a male specific bile acid, 3kPZS, and electrophysiologically discriminates it from other conspecific bile acids. This provides further physiological evidence to support the hypothesis that 3kPZS is a male pheromone (Li et al. 2002). It appears that in addition to conjugating groups at the carbon 3 and 24 positions, other functional groups at carbon 3 are also critical in determining the odor property of bile acids.
Abbreviations
- 3kACA:
-
3-keto allocholic acid
- 3kPZS:
-
3-keto petromyzonol sulfate
- ACA:
-
allocholic acid
- ANOVA:
-
analysis of variance
- ELISA:
-
enzyme-linked immunosorbent assay
- EOG:
-
electro-olfactogram
- PIR:
-
percent initial response
- PZS:
-
petromyzonol sulfate
- SMW:
-
spermiating male washings
References
Applegate VC (1950) Natural history of the sea lamprey (Petromyzon marinus) in Michigan. US Fish Wildl Serv Spec Sci Rep Fish Serv No 55
Bjerselius R, Li W, Teeter JH, Johnsen PB, Maniak PJ, Grant GC, Polkinghorne CN, Sorensen, PW (2000) Direct behavioural evidence that unique bile acids released by larval sea lamprey (Petromyzon marinus) function as a migratory pheromone. Can J Fish Aquat Sci 57:557–569
Caprio J, Byrd RP (1984) Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish. J Gen Physiol 84:403–422
Døving KB, Selset R, Thommesen G (1980) Olfactory sensitivity to bile acids in salmonid fishes. Acta Physiol Scand 108:123–131
Hara TJ (1994) The diversity of chemical stimulation in fish olfaction and gustation. Rev Fish Biol Fish 4:1–35
Hardisty MW, Potter IC (1971) The general biology of adult lampreys. In: Hardisty MW, Potter IC (eds) The Biology of lampreys, vol. 1. Academic Press, New York, pp 127–206
Haslewood GA, Tokes L (1969) Comparative studies of bile salts. Bile salts of the lamprey Petromyzon marinus L. Biochem J 114:179–184
Hellstrøm T, Døving KB (1986) Chemoreception of taurocholate in anosmic and sham- operated cod, Gadus morhua. Behav Brain Res 21:155–162
Jones KA, Hara TJ (1985) Behavioural responses of fishes to chemical cues: results from a new bioassay. J Fish Biol 27:495–504
Li W (1994) The olfactory biology of sea lamprey (Petromyzon marinus). PhD dissertation, University of Minnesota, St. Paul, USA
Li W, Sorensen PW (1997) Highly independent olfactory receptor sites for naturally occurring bile acids in the sea lamprey, Petromyzon marinus. J Comp Physiol A 180:429–438
Li W, Sorensen PW, Gallaher DD (1995) The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae. J Gen Physiol 105:567–587
Li W, Scott AP, Siefkes MJ, Yan H, Liu Q, Yun S-S, Gage DA (2002) Bile acid secreted by male sea lamprey that acts as a sex pheromone. Science 296:138–141
Michel WC, Lubomudrov LM (1995) Specificity and sensitivity of the olfactory organ of the zebrafish, Danio rerio. J Comp Physiol A 177:191–199
Moore HH, Schleen LP (1980) Changes in spawning runs of sea lamprey (Petromyzon marinus) in selected streams of Lake Superior after chemical control. Can J Fish Aquat Sci 37:1851–1860
Porkinghorne CN, Olson JM, Gallaher DD, Sorensen PW (2001) Larval sea lamprey release two unique bile acids to the water at a rate sufficient to produce detectable riverine pheromone plumes. Fish Physiol Biochem 24:15–30
Siefkes MJ, Bergstedt RA, Twohey MB, Li W (2003a) Chemosterilization of male sea lampreys does not affect sex pheromone release. Can J Fish Aquat Sci 60:23–31
Siefkes MJ, Scott AP, Zielinski B, Yun S-S, Li W (2003b) Male sea lampreys, Petromyzon marinus L., excrete a sex pheromone from gill epithelia. Biol Reprod 69:125–132
Sola C, Tosi L (1993) Bile acids and taurine as chemical stimuli for glass eels, Anguilla anguilla: a behavioral study. Environ Biol Fishes 37:197–204
Stabell OB (1987) Intraspecific pheromone discrimination and substrate marking by Atlantic salmon parr. J Chem Ecol 13:1625–1643
Vermeirssen ELM, Scott AP (2001) Male priming pheromone is present in bile, as well as urine, of female rainbow trout. J Fish Biol 1039–1045
Vrieze L, Sorensen PW (2001) Laboratory assessment of the role of a larval pheromone and natural stream odor in spawning stream localization by migratory sea lamprey (Petromyzon marinus). Can J Fish Aquat Sci 58:2374–2385
Yun S-S, Scott AP, Li W (2003) Pheromones of the male sea lamprey, Petromyzon marinus L: structural studies on a new compound, 3-keto allocholic acid, and 3-keto petromyzonol sulfate. Steroids 68:297–304
Yun S-S, Siefkes MJ, Scott AP, Li W (2002) Development and application of an ELISA for sea lamprey male sex pheromone. Gen Comp Endocrinol 129:163–170
Zhang C, Hara TJ (1994) Multiplicity of salmonid olfactory receptors for bile acids as evidenced by cross-adaptation and ligand binding assay. Chem Senses 19:579
Zhang C, Brown SB, Hara TJ (2001) Biochemical and physiological evidence that bile acids produced and released by lake char (Salvelinus namaycush) function as chemical signals. J Comp Physiol B 171:161–171
Acknowledgements
We thank Roger Bergstedt and the staff of US Geological Survey Hammond Bay Biological Station for accommodating us in their laboratory, and Michael Twohey and the staff of Marquette Biological Station for supplying us with sea lampreys. Dr. Sang-Seon Yun performed ELISA analysis and helped with bile acid preparation. Members of Li laboratory provided critique of an early version of this manuscript. The Great Lakes Fishery Commission financed this study. This research was approved by the Michigan State University, All University Committee on Animal Use and Care, and complied with all federal and state laws, policies, and rules for the humane use of laboratory animals in research.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Siefkes, M.J., Li, W. Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (Petromyzon marinus). J Comp Physiol A 190, 193–199 (2004). https://doi.org/10.1007/s00359-003-0484-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00359-003-0484-1