ORIGINS AND EVOLUTION OF BILE ACID PHEROMONES
By
Tyler John Buchinger

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Fisheries & Wildlife
2012

ABSTRACT

ORIGINS AND EVOLUTION OF BILE ACID PHEROMONES
By
Tyler John Buchinger

Fishes rely upon conspecific-released bile acids to attain information required for various
critical behaviors, the most commonly cited examples being migration and spawning. Numerous
investigations have elucidated the underlying physiology and behavior of fishes cueing to
conspecific bile acids, however the origins and evolution of such responses have yet to be
discussed. In Chapter 1, bile acids are presented as a model to investigate how natural and
sexual selection shape the design of aquatic chemical cues in a variety of contexts by
synthesizing relevant chemical, physiological, and behavioral literature and providing
hypotheses for contexts that have yet to be studied. In Chapter 2, I infer the origin of the sea
lamprey (Petromyzon marinus) bile alcohol mating signal, 3 keto petromyzonol sulfate, by
investigating the physiology and behavior of the ancestral silver lamprey (Ichthyomyzon
unicuspis). I provide evidence that the use of 3kPZS as a mating signal evolved as a result of a
sensory bias, whereas female preference evolved under natural selection rather than sexual
selection. The hypotheses and supporting data presented aid to fill gaps in evolution and
communication theory, for which the chemosensory modality is rarely considered.

ACKNOWLEDGMENTS

I would like to thank my advisors Dr. Nicholas Johnson and Dr. Weiming Li, and my
graduate committee, Dr. Janette Boughman and Dr. Michael Siefkes. The staff of the U.S.
Geological Survey Hammond Bay Biological Station patiently provided laboratory space and
guidance. The U.S. Fish and Wildlife Service Marquette Biological Station provided parasitic
lamprey species, and the Ludington Biological Station provided assistance with collecting nonparasitic lamprey species. I am thankful to the Li laboratory for support, especially Dr. Huiyong
Wang for performing LC-MS analysis and Dr. Yu-Wen Chung-Davidson and Anne Scott for
their assistance with histology. I am grateful for the hard work, suggestions, and friendship of the
following colleagues and technicians: C. Brant, T. Meckley, H. Kruckman, E. Buchinger, P.
Ganz, H. McMath, J. Yaklin, C. Dean, and E. Benzer. I am especially grateful to my wife
Elizabeth for her endless love, encouragement, and patience. I appreciate the financial support
provided by the Great Lakes Fishery Commission.

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TABLE OF CONENTS

LIST OF TABLES ......................................................................................................................... vi
LIST OF FIGURES ...................................................................................................................... vii
INTRODUCTION TO THESIS ..................................................................................................... 1
REFERENCES ............................................................................................................................ 4
CHAPTER 1
THE SWEET SMELL OF BILE; HOW, WHEN, AND WHY BILE ACIDS INFLUENCE FISH
BEHAVIOR .................................................................................................................................... 7
ABSTRACT ................................................................................................................................ 8
INTRODUCTION....................................................................................................................... 9
EVOLUTIONARY CONTEXT................................................................................................ 12
GENERAL FEATURES ........................................................................................................... 13
Metabolism ............................................................................................................................ 13
Structure ................................................................................................................................ 13
Function ................................................................................................................................. 14
RELEASE ................................................................................................................................. 15
Excretion ................................................................................................................................ 15
Environmental fate ................................................................................................................ 16
RESPONSE ............................................................................................................................... 18
Irritation ................................................................................................................................ 18
Individual recognition ........................................................................................................... 18
Foraging ................................................................................................................................ 19
Defense .................................................................................................................................. 20
Reproductive migrations........................................................................................................ 20
Sexual behaviors .................................................................................................................... 21
CONCLUSION ......................................................................................................................... 24
REFERENCES .......................................................................................................................... 26
CHAPTER 2
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ORIGIN OF A BILE ALCOHOL MATING SIGNAL IN AN ANCIENT AGNATHAN.......... 34
ABSTRACT .............................................................................................................................. 35
INTRODUCTION..................................................................................................................... 36
METHODS................................................................................................................................ 39
Experimental animals ............................................................................................................ 39
Release of bile acids .............................................................................................................. 39
Holding water and tissue collections ................................................................................. 39
Bile acid quantification ...................................................................................................... 41
Immunocytochemistry ........................................................................................................ 43
Behavioral response to 3kPZS............................................................................................... 43
Two-choice maze ................................................................................................................ 43
Quasi-natural stream ......................................................................................................... 44
RESULTS.................................................................................................................................. 47
Bile acid excretion by larvae ................................................................................................. 47
Bile acid biosynthesis and release by adults ......................................................................... 49
Behavioral response of adult female silver lamprey to 3kPZS .............................................. 56
DISCUSSION ........................................................................................................................... 60
REFERENCES .......................................................................................................................... 65

v

LIST OF TABLES

Table 2-1. Bile salts putatively involved in reproductive behavior of the sea lamprey
(Petromyzon marinus)……………………………………………………………………………42

vi

LIST OF FIGURES

Figure 2-1. Assays used to evaluate the behavioral responses of silver lamprey to 3kPZS. A)
Two-choice maze modified from Li et al. [2002] used to evaluate the proximate response of
silver lamprey to 3kPZS. The size was scaled down to half the size of that used for sea lamprey,
as silver lamprey are approximately half the size of sea lamprey. B) Quasi-natural stream used
to evaluate response of silver lamprey to 3kPZS in a natural setting. Large dashed lines represent
overhead-flow boards used to reduce turbulence. Small dashed lines represent mesh restricting
movement of lamprey. Arrows represent direction of flow. Dark bars represent PIT antennae
used to monitor movement of lamprey. Double angled-dashes represent breaks in the scale of the
large stream-like system, allowing a side-by-side comparison for the purposes of the figure. .... 46
Figure 2-2. Mean rate (ng/larva/hr ± standard error) at which silver lamprey larvae released 3
keto petromyzonol sulfate (3kPZS), petromyzonol sulfate (PZS), and petromyzonamine disulfate
(PADS) into the water. .................................................................................................................. 48
Figure 2-3. Immunopossitive staining (orange) to the 3-keto antibody in the liver tissue of
spermiated (A) and prespermiated (B) male silver lamprey. Scale-bar is 20µm. Both images
were taken using an X40 objective and the same exposure settings. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this thesis....................................................................................................................................... 50
Figure 2-4. Mean concentration of 3 keto petromyzonol sulfate (3kPZS; black), petromyzonol
sulfate (PZS; dark grey) and petromyzonamine disulfate (PADS; light grey; ng/g tissue ±
standard error) detected in the liver tissues of silver and sea lamprey, presented on a log scale. IF
Sil = immature female silver lamprey, MF SiL = mature female silver lamprey, IM SiL =
immature male silver lamprey, MM SiL = mature male silver lamprey, and MM SL = mature
male sea lamprey........................................................................................................................... 51
Figure 2-5. Histology of male silver lamprey gill tissues. H&E stained gill tissues of spermiating
males (A) show cells similar to previously documented glandular cells (large arrows). H&E
stained gill tissues of prespermiated males (B) do not show glandular cells. Immunoreactive
3kPZS (orange) in the gill tissue of spermiating (C) males was weak but localized in platelet
(small arrow) and glandular (large arrow) cells and in platelet cells (small arrow) of
prespermiating (D) males. Scale-bar is 20µm. Both images were taken using an X40 objective
and the exposure settings. ............................................................................................................. 53
Figure 2-6. Mean concentration of 3 keto petromyzonol sulfate (3kPZS; black) and
petromyzonol sulfate (PZS; dark grey; ng/g tissue ± standard error) detected in the gill tissues of
silver lamprey, presented on a log scale. IF Sil = immature female silver lamprey, MF SiL =

vii

mature female silver lamprey, IM SiL = immature male silver lamprey, MM SiL = mature male
silver lamprey, and MM SL = mature male sea lamprey. NA = samples not able to be taken. .. 54
Figure 2-7. Mean release (ng/g individual/hr) of 3 keto petromyzonol sulfate (3kPZS) into the
water as quantified with UPLC-MS/MS, presented on a log scale. IF Sil = immature female
silver lamprey, MF SiL = mature female silver lamprey, IM SiL = immature male silver lamprey,
MM SiL = mature male silver lamprey, and MM SL = mature male sea lamprey....................... 55
Figure 2-8. Near-source preference of silver lamprey of each sex and maturity combination to 3
keto petromyzonol sulfate (3kPZS) as evaluated using a two-choice maze. Error bars represent
the standard error of the mean. The response of ovulated female sea lamprey to 3kPZS is shown
for reference (N. Johnson, unpublished data). The asterisk represents significance as determined
using a Wilcoxon signed rank test. IF Sil = immature female silver lamprey, MF SiL = mature
female silver lamprey, and MF SL = mature female sea lamprey. ............................................... 57
Figure 2-9. Near-source behavior of migratory and spawning female silver lamprey and
spawning sea lamprey in the quasi-natural stream experiments. Data are presented as the mean
proportion of females that entered the 3 keto petromyzonol sulfate (3kPZS) baited nest (dark
grey) or the methanol baited nest (light grey). Error bars represent the standard error of the mean.
Significant differences between the response to 3kPZS and response to methanol (α = 0.05) as
determined using logistic regression are displayed with an asterisk. N/N = the number of lamprey
that enter the odor/ total number of lamprey that made the choice between odors. IF Sil =
immature female silver lamprey, MF SiL = mature female silver lamprey, and MF SL = mature
female sea lamprey. ...................................................................................................................... 58
Figure 2-10. Upstream movement of migratory and spawning female silver lamprey and
spawning sea lamprey in quasi-natural stream experiments. Data are presented as the mean
proportion of females that moved upstream when 3 keto petromyzonol sulfate (3kPZS; dark
grey) or methanol (light grey) was applied. Error bars represent the standard error of the mean.
Significant differences (α = 0.05) between the response to 3kPZS and the response to methanol
as determined using logistic regression are displayed with an asterisk. N = the number of
lamprey that move upstream/total number of lamprey released. IF Sil = immature female silver
lamprey, MF SiL = mature female silver lamprey, and MF SL = mature female sea lamprey. ... 59

viii

INTRODUCTION TO THESIS

Fishes rely upon abiotic and biotic olfactory cues to facilitate behaviors critical for
foraging, defense, migration, and reproduction [1]. The olfactory epithelium of fishes is acutely
and specifically tuned to detect prostaglandins, steroid hormones, amino acids, and bile acids [2],
each of which has been shown to influence the fish behavior [3, 4, 5]. The suite of olfactory cues
influencing fish behavior raises questions regarding the evolutionary mechanisms responsible.
Theoretical considerations regarding the evolution of the involvement of prostaglandins and
steroid hormones in chemical cueing have been discussed [6, 7, 8]. Consideration of the
evolution of chemoreception and its application to communication theory is generally lacking
[9]. I aim to fill a portion of this gap in the chemoreception literature, providing first a theoretical
background for the selective pressures on chemical cues, and second an empirical test of the
evolutionary mechanism leading to pheromone systems, in each case using bile acids as a model.
Chapter 1, titled “The sweet smell of bile; how, when, and why bile acids influence fish
behavior”, reviews biochemical, physiological, and ecological literature pertinent to the
evolution of bile acids as chemical cues, and proposes further hypothesis regarding when
selection will favor decisions based upon olfactory detection of bile acids. Hypotheses regarding
the biological significance of the olfactory detection of bile acids range from behaviors
associated with foraging, reproductive migrations, and sexual communication, and include fishes
from all life history strategies and points in vertebrate evolution. The suitability of bile acids as
informative chemical cues is evaluated on many fronts, including bile acid structure and
function, release into the environment, detection, and behavioral influence.

1

Chapter 2, titled “Origin of a bile alcohol mating signal in an ancient Agnathan”, infers
the evolution of a bile alcohol mating pheromone by comparing the behavior and physiology of
the sea lamprey (Petromyzon marinus) and the ancestral silver lamprey (Ichthyomyzon unicuspis)
[10]. The sea lamprey has recently become a model species to study chemical communication
[11]. Bile acids emanating from stream-resident larvae direct adults from lakes or the ocean into
streams with a recent history of high offspring survival [12]. Upon sexual maturation, males
release large amounts of 7α, 12α, 24-trihydroxy-5α -cholan-3-one-24-sulfate (3 keto
petromyzonol sulfate, 3kPZS) via glandular cells in the gills [5, 13], which is a potent attractant
of sexually mature females [14, 15]. Bile alcohols likely hold very little inherent information as
to the reproductive fitness of an individual, thus I hypothesized that the female preference for
3kPZS evolved via a sensory bias of females [16]. I hypothesized that the ancestral silver
lamprey cue onto larval-released 3kPZS to locate streams containing habitat conducive to
offspring survival and that adult silver lamprey males have not adapted to manipulate such
preference by signaling with 3kPZS. As predicted in silver lamprey, (a) 3kPZS was found in
high concentration in larval holding waters, (b) more migratory females move upstream in
response to 3kPZS, and spawning-phase females retain the upstream movement response to
3kPZS, and (c) spawning-phase males do not biosynthesize or release 3kPZS or similar bile
acids at rates likely to be detected by females. Based upon the above findings, I infer that the
female preference for 3kPZS evolved via natural selection in silver lamprey, but male signaling
evolved later in the sea lamprey.
The information presented in this thesis broadens the understanding of chemical cueing,
by filling gaps in the literature pertaining to the evolution of bile acid communication systems.
Considerations of the evolutionary mechanisms involved in chemical communication strengthens

2

hypotheses regarding identities of the compounds involved, and aids in understanding how
animals exchange information. An understanding of pheromones is useful for basic [9] and
applied [17] research objectives.

3

REFERENCES

4

REFERENCES

1. Hara TJ. 1975. Olfaction in fish. Progress in Neurobiology 5: 271–335.
2. Hara TJ. 1994. The diversity of chemical stimulation in fish olfaction and gustation.
Reviews in Fish Biology and Fisheries 4: 1–35.
3. Stacey N, Chojnacki A, Narayanan A, Cole T, et al. 2003. Hormonally derived sex
pheromones in fish: exogenous cues and signals from gonad to brain. Canadian Journal of
Physiology and Pharmacology 81: 329–41.
4. Hara T. 2006. Feeding behaviour in some teleosts is triggered by single amino acids
primarily through olfaction. Journal of Fish Biology 68: 810–25.
5. Li W, Scott AP, Siefkes MJ, Yan H, et al. 2002. Bile acid secreted by male sea lamprey that
acts as a sex pheromone. Science 296: 138-41.
6. Sorensen PW, Scott AP. 1994. The evolution of hormonal sex pheromones in teleost fish:
poor correlation between the pattern of steroid release by goldfish and olfactory sensitivity
suggests that these cues evolved as a result of chemical spying rather than signal
specialization. Acta Physiologica Scandinavica 152: 191–205.
7. Sorensen PW, Stacey NE. 1999. Evolution and specialization of fish hormonal pheromones.
Advances in Chemical Signals in Vertebrates : 15–47.
8. Appelt CW, Sorensen PW. 2007. Female goldfish signal spawning readiness by altering
when and where they release a urinary pheromone. Animal Behaviour 74: 1329–38.
9. Steiger S, Schmitt T, Schaefer HM. 2011. The origin and dynamic evolution of chemical
information transfer. Proceedings of the Royal Society B: Biological Sciences 278: 970–9.
10. Potter IC, Gill HS. 2003. Adaptive radiation of lampreys. Journal of Great Lakes Research
29: 95–112.
11. Li W. 2005. Potential multiple functions of a male sea lamprey pheromone. Chemical Senses
30: i307–8.
12. Sorensen PW, Hoye TR. 2007. A critical review of the discovery and application of a
migratory pheromone in an invasive fish, the sea lamprey Petromyzon marinus L. Journal of
Fish Biology 71: 100–14.
13. Siefkes MJ, Scott AP, Zielinski B, Yun SS, et al. 2003. Male sea lampreys, Petromyzon
marinus L., excrete a sex pheromone from gill epithelia. Biology of Reproduction 69: 125-32.

5

14. Siefkes MJ, Winterstein SR, Li W. 2005. Evidence that 3-keto petromyzonol sulphate
specifically attracts ovulating female sea lamprey, Petromyzon marinus. Animal Behaviour
70: 1037–45.
15. Johnson NS, Yun SS, Thompson HT, Brant CO, et al. 2009. A synthesized pheromone
induces upstream movement in female sea lamprey and summons them into traps.
Proceedings of the National Academy of Sciences 106: 1021–26.
16. Endler JA, Basolo AL. 1998. Sensory ecology, receiver biases and sexual selection. Trends
in Ecology & Evolution 13: 415–20.
17. Li W, Twohey M, Jones M, Wagner M. 2007. Research to guide use of pheromones to
control sea lamprey. Journal of Great Lakes Research 33: 70–86.

6

CHAPTER 1

THE SWEET SMELL OF BILE; HOW, WHEN, AND WHY BILE ACIDS INFLUENCE FISH
BEHAVIOR

7

ABSTRACT

The increasingly diverse physiological capacities of bile acids have recently been
augmented by new functional hypotheses pertaining to the sensitive tuning of the fish olfactory
epithelium to detect bile fluids of other fishes. Hypotheses regarding the biological significance
of the olfactory detection of bile acids range from behaviors associated with foraging,
reproductive migrations, and sexual communication, and include fishes from all life history
strategies and points in vertebrate evolution. I present bile acids as a model to investigate how
natural and sexual selection shape the characteristics of aquatic chemical cues in a variety of
contexts. Theoretical considerations regarding the selective pressures influencing the identity of
chemical cues are briefly summarized. The suitability of bile acids as informative chemical cues
is traced by synthesizing literature on bile acid structure and function, release into the
environment, detection, and behavioral influence. Hypotheses pertaining to the contexts in which
bile acids are effective in conveying information between fishes are presented.

8

INTRODUCTION

Describing the physiology, pathology, and evolution of bile salts has been a focal point of
biologist for over a century. Physiologists have described the primary functions of bile acids in
mammals to include cholesterol elimination, dietary lipid solubilization, stimulation of bile flow
and phospholipid secretion, and regulation of bile acid and cholesterol synthesis [1]. Physicians
study errors in the enterohepatic pathway of bile acids resulting in disruption of cholesterol
metabolism, and disease of the liver and intestinal tract [2]. Understanding of bile acid biology
has given direction towards potential therapeutics, providing insights into medical concerns such
as obesity [3] and biliary artesia [Yeh et al. submitted]. Chemists have found great interest in bile
acids, specifically in the systematic variation of bile salt structure, which seems to follow
vertebrate evolution [4]. The broad interest in bile acids allows for an integrative understanding
of the biology of an entire class of compounds.
An emerging field of bile acid biology is the olfactory detection and associated
behavioral response in fishes. Doving and colleagues [5] first postulated that bile acids
emanating from stream resident conspecifics direct anadromous Arctic char (Salvelinus alpinus)
to natal streams in which to spawn. Now, after three decades of research into biliary odors, it
seems the olfactory function of bile acids may be more widespread than that of any other biomolecule. While amino acids and sex hormones (prostaglandins and steroid hormones) are
hypothesized to function as food odors [6] and mating cues in freshwater teleosts [7],
respectively, the hypotheses of bile acid cues extend to a much larger array of contexts.
Olfactory detection of bile acids has been proposed to influence behaviors ranging from foraging
aggregations [8] to sexual signaling [9]. Olfactory detection of bile acids evolved early in the

9

vertebrate lineage [10] and has been conserved in more modern fishes [11]. Hypotheses of fishes
utilizing bile acid cues include species employing a number of different life histories, including
marine [12], freshwater [13], anadromy [10], and catadromy [8].
The most comprehensive examination of bile acid cueing focuses on the sea lamprey
(Petromyzon marinus). Sea lamprey cue onto the odor of stream-resident larvae during their
upstream spawning migrations, the active components of which have been suggested to be
petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and the bile alcohol
petromyzonol sulfate (PZS) [10, 14]. A second bile alcohol, 3 keto petromyzonol sulfate
(3kPZS) has a minor function in directing migrating female sea lamprey upstream to spawning
grounds (Brant et al. submitted), and acts as a priming pheromone in males (Chung-Davidson et
al. in prep). Sexually mature females are strongly attracted to 3kPZS, which is released via
specialized glandular in the gills of mature males [9, 15]. Conspecific bile acids may also
indicate suitable feeding habitat to sea lamprey in the prolarval stage [16].
Investigation of the olfactory detection of bile acids not only contributes to a
comprehensive understanding of their biological function, but also provides a useful model to
study the evolution and characteristics of chemical signals. The variety of contexts in which
fishes have adapted to cue on bile acids allows for a discussion on the trajectory of chemical
signal design as shaped by both natural and sexual selection. Several authors have briefly
proposed specific characteristics of bile acids giving them selective advantage as chemical cues,
suggesting that structural diversity [5] and efficient synthesis [17] are important features.
However, with the extent of bile acid chemical cueing now becoming apparent, further
consideration of bile acid characteristics with respect to chemical communication is warranted.
Here, I expand upon previous discussions of the selective advantages to biliary cueing by

10

synthesizing pertinent biochemical, physiological, and ecological literature and providing further
hypotheses regarding the contexts in which bile acids are favored as olfactory cues. Suitability of
bile acids as informative chemical cues is traced from general features, release into the
environment, and subsequent detection and response of receivers.

11

EVOLUTIONARY CONTEXT

Understanding the selective advantages of biliary cueing first requires a theoretical
consideration of the evolution of aquatic odors. The aquatic environment hosts a plethora of
chemicals, originating from conspecific and heterospecific fish, other biota, and the surrounding
habitat. The olfactory system of fish has incredible capability to discriminate between the
molecules reaching the olfactory epithelium, including those unfamiliar to an individual [18].
Odors that are informative become cues when individuals show higher fitness in association with
an increased response to a compound [19]. A change in behavior based upon unaltered release of
a compound has been termed spying [20]. The efficacy of spying in acquiring information
depends upon the rate at which a compound emanates from the releaser, the number of releasers,
and the active space needed. Active communication may evolve as a result of receiver biases,
where the release of a compound is manipulated to influence the behavior of receivers possessing
a pre-existing response [20] (Chapter 2). However, in situations where the cue contains inherent
information, active communication may evolve in response to mutual direct benefits. The design
of chemical signals depends not only on the pre-existing preference of the receiver, but also upon
the efficiency of production, release, transmission, and reception [21].

12

GENERAL FEATURES

Metabolism
As the end result of cholesterol catabolism, bile acids are metabolically inexpensive to
synthesize in the quantities required to be perceived by other fish. Approximately 90% of
cholesterol, accumulated through dietary intake or through biosynthesis from acetyl CoA [22], is
catabolized into bile acids in the hepatocyte [2]. Following conjugation with a sulfate (bile
alcohols) or glycine, taurine, or a taurine derivative (bile acids), bile salts are secreted, via bile
fluid, into the intestine. The enterohepatic circulation of bile acids is extremely efficient; humans
reabsorb and recycle approximately 95% of the bile acids secreted into the intestine [23]. In
comparison, only 5% of cholesterol is converted to steroid hormones. The biosynthetic
differences between bile acids and steroid hormones may be a factor leading to the evolution of
bile acid sex pheromones used by species reproducing in high-volume environments, such as the
sea lamprey [9, 17].

Structure
The patterned variability in bile acid structure is a unique feature providing high potential
for specificity. Bile acids appear to be an adaptation of vertebrates, hence any bile acids present
in aquatic environments can be linked to fish. Across vertebrate classes (fish, amphibians,
reptiles, birds, and mammals), bile salts show a progression from C27 bile alcohols to C24 bile
acids, with some groups having C27 bile acids as intermediates [4]. Dominant bile salts differ
between fish taxa; for example, Elasmobranchii (sharks, skates, and rays) primarily produce a
default C27 bile alcohol, compared to the C24 bile acids frequently used by Actinopterygii (ray-

13

finned fish) and the C24 bile alcohols of Petromzyontiformes [24]. Bile salt profiles show
substantial variation between orders of fishes, and occasionally between closely related species
[24]. Seemingly minor variation in bile acid structure likely increase the specificity of odors
substantially, as olfactory receptors are acutely sensitive to structural differences. Sea lamprey
differentiate between the migratory odor PZS and spawning odor 3kPZS, with separate receptors
binding each ligand based upon the presence of a 3-keto group [25].

Function
The physiological functions of bile acids, likely existing prior to olfactory functions [20],
align with requirements for effective and informative odors. The primary function of bile acids
and alcohols is the solubilization of dietary fats in the intestine; however, bile salts fulfill many
other critical physiological functions (e.g. fluid regulation, ion transport, cholesterol elimination)
[2]. The function and subsequent excretion of bile acids is relatively unchanging compared to
steroid hormones, for example, which primarily emanate from individuals of a specific
reproductive status. Such consistency in function provides ample opportunity for fishes to
establish an association between an event and the odor of bile. Several capacities of bile acids are
particularly relevant to aquatic olfaction. The primary emulsifying function of bile acids requires
solubility [26]. Solubility is also a major requirement for aquatic olfaction, likely as important as
volatility is for terrestrial olfaction [19]. Bile acids have recently been recognized as endocrine
signaling molecules [27]. Intraspecific signaling likely pre-adapts bile acids to serve as
interspecific cues, requiring only the mutation externalizing receptors [28].

14

RELEASE

Excretion
A portion of the bile acid pool is lost as metabolic waste, eventually reaching the
olfactory epithelium of nearby individuals. Most bile acids are cleared of the intestine via feces
[13, 29], although exceptions include the release of a bile acids via gills by sea lamprey [15] and
urine of lake trout [30]. Trace amounts of bile salts have been found in the urine of rainbow trout
[31]. Marine teleosts likely excrete bile salts as a result of osmoregulation, via the constant flow
of fluids through the intestine [32, 33]. In comparison, amino acids escape via mucus [34, 35],
sex hormones are excreted via gills [36], and both are commonly excreted through urine [31, 37]
and gonadal fluids [38, 39, 40].
The bile acids released into the environment are also those most relevant to olfaction.
Many of the excreted bile acids are sulfated, whereas sulfation decreases intestinal permeability
and may be an adaptive mechanism for excreting excess bile salts [41]. In the aquatic
environment, conjugation with a sulfate increases solubility and often yields stronger stimulation
of the olfactory epithelium [11, 25, 42]. The sulfate group on known pheromones is crucial for
behavioral activity; for example, the lack of the sulfate group voids the behavioral influence of
3kPZS on ovulated female sea lamprey [43].
Although critically important in considering odors, the release rates of metabolites are
extremely variable. A portion of release variability can be explained by the physiological and
reproductive contexts. Bile salt and amino acid release into the water changes with diet in sea
lamprey [29] and goldfish [35], respectively. Release rates can drastically change across life
stage and sex. Sea lamprey release approximately 30 ng/g fish/h of a bile alcohol migratory cue

15

as juveniles [44], but later spawning males release approximately 1000 ng/g fish/h of a related
bile alcohol sex signal [9].

Environmental fate
Bile salts must be structurally stable to be effective in digestion, and likewise to be
effective chemical cues [26, 19]. Bile salts are extremely resistant to digestive enzymes within an
organism [26]. Further in the gastrointestinal tract, resident bacteria deconjugate bile salts, and
once excreted, degrade steroids outside of the organism [45, 46]. However, oxidation of
hydroxyl groups and reduction of carboxyl groups occur quite slow [47]. Relative to when the
information will be acquired by the receiver, bile salts do not degrade quickly enough to inhibit
any function as aquatic cues. For example, lamprey-released 3kPZS in river water samples does
not degrade dramatically until about four days later [48]. Again, sea lamprey larval odors have a
half-life of approximately three days [44]. Degradation at this rate is inconsequential, the odors
in the stream would either have been replenished, or the information would no longer be relevant
as the releaser has moved. In comparison, the half-life of some goldfish-released sex steroids is
about 6 hours [49].
The structural variability of bile salts gives rise to compounds with extremely diverse
properties, allowing them to act as cues in many contexts. Many reproductive odors need to
function instantly. In this case, an odor that is extremely stable will eventually provide false
information. Some behaviors require a cue that functions over a longer period of time. Bile salts
have been shown to function in this scenario as well, as some are absorbed by organic detritus or
adhesive to substrate [5]. Lake trout may be an example of a fish using bile salts as such a
marker. Lake trout spawn over reefs in the fall, with young rising up to feed the following

16

summer [50]. Adult males, the first to return for spawning, exhibit cleaning behaviors prior to
spawning, and the cleaned area is the eventual site of the spawning act [51]. The bile salts of
young lake trout may be used by adults to locate spawning reefs [52, 30, 13]. Male cleaning
behavior may effectively bring up bile salts that have been absorbed by detritus in the months
since deposition. Atlantic salmon may also use less water-soluble bile acids to mark substrate
[53]. Adhesion to substrate or organic matter has not been shown to interfere with the sensitivity
of fish towards bile acids [44].

17

RESPONSE

Irritation
Pre-existing stimulation, such as irritation, may have given evolutionary preference to
bile acid odors. Evolution of an odor into a chemical cue begins with general olfactory
stimulation [20]. Although fish can detect countless odors, some compounds may have inherent
properties that increase stimulation of the olfactory epithelium. Bile acids are potent stimulants
of olfactory receptors, but stimulation may be a result of many fishes using bile salts as chemical
cues, rather than any inherent value of bile salts [54]. Bile salts may cause general irritation,
potentially resulting from their strong detergent properties. Doving et al. [5] first discussed the
possibility of bile salts as strong acids increasing stimulation on the olfactory epithelium, but
resolved the hypothesis unlikely since amino acid receptors did not reveal the same stimulation
as bile acids. However, Erickson and Caprio [55] found a portion of the electrical response of
channel catfish (Ictalurus punctatus) to bile salts could not be attributed to the olfactory neural
activity, indicating simple irritation. Sensory bias theory of signal design predicts that signals
which are inherently conspicuous to the sensory system of receivers will be evolutionarily
favored. In the context of olfaction, inherent conspicuity could originate from properties causing
irritation, which may have shifted the trajectory of chemical signal design towards bile acids.

Individual recognition
The detection of many bile acids is not species-specific, potentially reflecting a
mechanism for detecting proximate individuals regardless of species [56]. Taurocholic acid is
detected with acute sensitivity by all species investigated, including trout (Salvelinus sp.) [13, 5],

18

eels (Anguilla anguilla) [56], goldfish (Carassius auratus) [57], and sea lamprey [10]. The
production of taurocholic acid is also common across many fish species, including lake trout
[13], sol (Solea senegalensis) [12], and eels [8]. The general detection of bile acids may even
extend to those not produced by conspecifics [56]. Non-specific detection and production of bile
acids may provide fishes with an effective way to detect nearby individuals in an environment
where vision is often unreliable. Detection of taurocholate may be a mechanism to recognize
teleost fishes, an ability that would likely be advantageous to all fishes, as teleosts are the most
abundant taxa of fishes.

Foraging
As the release of bile acids coincides with metabolic processes, detection of conspecific
bile fluids may confer information regarding foraging opportunities. While amino acids suggest
food may be present [54, 6], bile salts, released as a result of digestion and influenced by diet,
could indicate that successful feeding is occurring. Unlike amino acids, bile acids may confer
relatively specific information regarding foraging success of conspecifics, considering the
patterns of bile salt structure discussed earlier [4]. Specific foraging information may be
particularly important to fishes undergoing extensive foraging migrations. Catadromous glass
eels (Anguilla rostrata and A. anguilla) have been hypothesized to cue onto conspecific bile
during migration into streams to forage [58, 8]. Attraction to the scent of conspecific bile acids is
concentration-dependent, possibly indicating a mechanism to detect systems which are at
capacity [8]. Given the great investment of long-distance migrations, individuals likely face a
substantial decline in fitness with incorrectly choosing a system lacking suitable foraging
opportunity. Although amino acids are likely suitable in conferring immediate foraging

19

opportunities, fishes investing in long-distance foraging migrations likely require more specific
information.

Defense
While the identity of alarm cues remains elusive [59], it is interesting to speculate as to
the role of bile salts in anti-predator behaviors [56]. The alarm response of some fish is
dependent upon the recent diet of the predator [60]. Although the synthesis of primary bile salts
is not thought to be influenced by diet, the secretion of bile salts into the intestine and leaking
into the water are effected by diet [29, 44]. Bacteria ingested may also change with diet, which
results in different secondary bile salts. Some species of insects use bacteria modified
compounds as cues, thus it seems reasonable that secondary bile salts produced by bacteria living
on certain prey species may also be alarm cues to potential prey [61].

Reproductive migrations
High-investment reproductive migrations may also be directed by bile acids emanating
from stream-resident conspecifics [5, 10]. The hypothesis seems plausible, as the bile fluid of
stream-residents specifically indicates offspring survival and productivity. Experimental
evidence is supportive; for example, Arctic char are acutely sensitive to bile acids [5], show
behavioral preference for conspecific bile acids [62], and return to the stream containing
conspecific odors [63]. However, contrasting empirical evidence indicates some fishes imprint
on unique amino acids profiles of the natal stream. Masu salmon (Onchorhynchus masou) are
acutely sensitive to amino acids mixtures matching that of the natal stream [64], and show
behavioral preference for the mixture [65]. Two highly debated hypotheses have emerged from

20

studies on mechanisms of reproductive migrations: the imprinting hypothesis [66] and the
pheromone hypothesis [63]. The imprinting hypothesis suggests that streams have a unique odor,
learned during the seaward migration of smolts [66]. The pheromone hypothesis suggests that
returning adults possess an innate recognition of a specific odor of stream-resident conspecifics
[63].
Examining the phylogeny of fishes undergoing reproductive migrations may provide an
explanation for the mechanistic differences. One hypothesis is that the use of bile acids to guide
migrations may be an ancestral trait, and that homing in on amino acid profiles is a more recent
adaptation. Lampreys, likely the earliest anadromous fishes, locate spawning streams following
bile acids emanating from stream-resident larvae [10, 67]. Early salmonid genera also locate
spawning sites using conspecific bile acids (Arctic char) [5, 68]. Imprinting upon amino acid
profiles may have evolved in intermediate genera of salmonids. Early Onchorhynchus species
imprint on amino acids and cue onto conspecific bile acids (Sockeye salmon, O. nerka) [69, 70].
Modern Onchorhynchus species rely only upon imprinting (Masu salmon) [64, 65, 71].
Ecologically, the scheme makes sense; the presence of conspecifics is a basic yet reliable
indicator of suitable habitat, but not as specific as learned amino acid profiles.

Sexual behaviors
Research into sex odors has yielded great diversity of origins; amino acids [38, 72], bile
salts [9], and sex hormones [73, 7] have each been documented as sex odors in fish. The use of
hormonal pheromones is not surprising, given the release of hormone metabolites often indicates
the sex and reproductive status of the releaser [28, 7]. Amino acids and bile salts may also
indicate the status of the releaser, as a result of diet changes associated with reproduction,

21

osmoregulatory changes associated with diadromy [32], or the breakdown of follicular cells [38].
Compounds likely evolve into sexual cues based upon the information that is inherent with their
passive leaking [20]. However, information stemming from change in bile acid or amino acid
release is quite vague in comparison to sex hormones, and indeed many of the fish investigated
to date use sex hormones to mediate spawning behaviors [7]. Nevertheless, some fish have
evolved to utilize bile acids as sex pheromones. The deviation of such cases can likely be
explained by the life history of the fish, and the specific behaviors being mediated by conspecific
odors.
Environmental differences facing marine fish resulting in contrasting osmoregulation
apply a new set of selective pressures pertaining to chemical communication, possibly favoring
the use of bile acids as chemical cues rather than hormones [32]. The majority of the studies
investigating sexual cues in fish have focused on freshwater teleosts. Freshwater teleosts produce
large amounts of urine, constantly leaking sex hormones into the water [37]. Marine teleosts
produce very little urine, but rather constantly excrete an intestinal fluid which contains bile
acids. The few studies completed on marine teleosts support the hypothesis that bile acids cues
are favored over hormonal cues [12, 32]. Sex hormones are released by European eels at minimal
rates and are not stimulatory to the olfactory epithelium [74]. Bile salts released by eels are likely
more informative regarding mate selection, as they change with maturity and are potent olfactory
stimulants [33, 56].
While leaking hormones are useful cues for near-source sexual behaviors, they are likely
not suitable to mediate long distance sexual behaviors. The inherent leaking of metabolic bile
salts and amino acids may also be unfit as long distance sex odors. Some sexual behaviors, such
as that of the sea lamprey, require an odor to direct an individual a long distance to mates. The

22

distance presents a problem with spying; migratory fish often cue based upon the amino acids or
bile salts of an entire system or population of fish, while long distance sexual behavior often
requires fish to home in on an individual mate. This may be an underlying pressure favoring bile
acids as chemical signals over long distances and high flows. Increasing the release rate of
hormones to produce effective concentrations would be metabolically expensive, while the
efficient synthetic pathway of bile salts allows them to function as inexpensive long distance
signals [9].
In cases where leaking of metabolites is inapt to cue conspecifics, signals may evolve
based on the existing preference of the receiver [20]. The sea lamprey and the Masu salmon
provide excellent contrasting examples of the use of bile acid and amino acid signals. Migratory
sea lamprey locate spawning streams following the bile acid plume of stream-resident
conspecifics [14]. Masu salmon home in on their natal stream following a stream-specific amino
acid profile [64]. The male sea lamprey and the female Masu salmon arrive at the spawning site
prior to the opposite sex, and begin nest construction. The long-distance movement of female sea
lamprey and male Masu salmon to the newly-constructed nest could only be directed by a signal
released at high rates. In each case, the signal matches the receiver’s bias; male sea lamprey
signal females to the nest by releasing a bile alcohol at high rates and female Masu salmon signal
males to the nest by releasing an amino acid at high rates [9, 72].

23

CONCLUSION

In an environment where most sensory modalities are unreliable, bile acids seem to be a
consistent information source for fishes of numerous life histories, making decisions pertaining
to proximate individuals, foraging opportunity, predatory threats, rearing habitat, and suitable
mates. The olfactory detection of bile acids is useful to discuss the dynamic evolution of odors.
The irritative detergent properties of bile acids may create a bias in the sensory systems of fish.
Such biases are amplified via natural selection when information pertaining to foraging
opportunity, predation, or productive habitat is gathered through the detection of bile acids. The
advantage of acquiring such information from bile acids can be explained by considering
previous discussions of efficient signals, specifically highlighting signal generation,
transmission, and detection [21]. Bile acids seem to match theoretical ideals for chemical
signaling in various contexts; consistent, efficient, and meaningful generation, taxa specific
structural variation, and transmission appropriate for long or short term chemical cues. Finally,
the many behavioral responses shaped by the information inherent to bile acids create a receiver
bias which may be manipulated by contexts requiring active signaling.
The vast diversity of detection of bile acids and associated behavioral responses results in
expansive knowledge gaps as to the biological function. While supporting electrophysiological
evidence abounds, data on bile acid release and subsequent behavioral changes in conspecifics is
relatively lacking. Nearly all studies on the olfactory detection of bile acids, or other compounds,
have examined freshwater teleosts, with a small number of recent studies on marine teleosts.
Studies directed at a broader variety of species, such as marine and non-teleost fish, would shed
light on the selective pressures influencing the design of chemical signals.

24

Although the olfactory detection of bile acids likely does not represent a truly “sweet”
smell, the scent likely does deliver information crucial to fishes making decisions. The
widespread olfactory detection and behavioral influence of bile acids offers a useful model
system to discuss the gathering of chemical information by fish. Bile acids will continue to
capture the attention of researchers from all realms of study, including medicine [Yeh et al.
submitted], evolution [4], ecology [9], and natural resource management [75].

25

REFERENCES

26

REFERENCES

1. Hofmann AF. 1999. The continuing importance of bile acids in liver and intestinal disease.
Archives of Internal Medicine 159: 2647–58.
2. Hofmann AF, Hagey LR. 2008. Bile acids: chemistry, pathochemistry, biology,
pathobiology, and therapeutics. Cellular and Molecular Life Sciences 65: 2461–83.
3. Watanabe M, Houten SM, Mataki C, Christoffolete MA, et al. 2006. Bile acids induce
energy expenditure by promoting intracellular thyroid hormone activation. Nature 439:
484–9.
4. Hofmann AF, Hagey LR, Krasowski MD. 2010. Bile salts of vertebrates: structural
variation and possible evolutionary significance. Journal of Lipid Research 51: 226–46.
5. Doving KB, Selset R, Thommesen G. 1980. Olfactory sensitivity to bile acids in salmonid
fishes. Acta Physiologica Scandinavica 108: 123–31.
6. Hara T. 2006. Feeding behaviour in some teleosts is triggered by single amino acids
primarily through olfaction. Journal of Fish Biology 68: 810–25.
7. Stacey N, Chojnacki A, Narayanan A, Cole T, et al. 2003. Hormonally derived sex
pheromones in fish: exogenous cues and signals from gonad to brain. Canadian Journal
of Physiology and Pharmacology 81: 329–41.
8. Sola C, Tosi L. 1993. Bile salts and taurine as chemical stimuli for glass eels, Anguilla
anguilla: a behavioural study. Environmental Biology of Fishes 37: 197–204.
9. Li W, Scott AP, Siefkes MJ, Yan H, et al. 2002. Bile acid secreted by male sea lamprey that
acts as a sex pheromone. Science 296: 138–41.
10. 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. The Journal of General Physiology 105: 569–87.
11. Michel W, Lubomudrov L. 1995. Specificity and sensitivity of the olfactory organ of the
zebrafish, Danio rerio. Journal of Comparative Physiology A: Neuroethology, Sensory,
Neural, and Behavioral Physiology 177: 191–9.
12. Velez Z, Hubbard PC, Welham K, Hardege JD, et al. 2009. Identification, release and
olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (Solea
senegalensis). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural,
and Behavioral Physiology 195: 691–8.

27

13. 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. Journal of Comparative Physiology B: Biochemical, Systemic, and
Environmental Physiology 171: 161–71.
14. Sorensen PW, Fine JM, Dvornikovs V, Jeffrey CS, et al. 2005. Mixture of new sulfated
steroids functions as a migratory pheromone in the sea lamprey. Nature Chemical
Biology 1: 324–8.
15. Siefkes MJ, Scott AP, Zielinski B, Yun SS, et al. 2003. Male sea lampreys, Petromyzon
marinus L., excrete a sex pheromone from gill epithelia. Biology of Reproduction 69:
125–32.
16. Zielinski BS, Fredricks K, McDonald R, Zaidi AU. 2005. Morphological and
electrophysiological examination of olfactory sensory neurons during the early
developmental prolarval stage of the sea lamprey Petromyzon marinus L. Journal of
Neurocytology 34: 209–16.
17. Li W. 2005. Potential multiple functions of a male sea lamprey pheromone. Chemical Senses
30: i307–8.
18. Kleerekoper H. 1969. Olfaction in Fishes. Indiana University Press.
19. Wyatt TD. 2003. Pheromones and Animal Behaviour. Cambridge: Cambridge University
Press.
20. Sorensen PW, Stacey NE. 1999. Evolution and specialization of fish hormonal pheromones.
Advances in Chemical Signals in Vertebrates: 15–47.
21. Endler JA, Basolo AL. 1998. Sensory ecology, receiver biases and sexual selection. Trends
in Ecology & Evolution 13: 415–20.
22. Liscum L. 2008. Cholesterol biosynthesis. In Biochemistry of Lipids, Lipoproteins and
Membranes (Fifth Edition). Elsevier. p 399–XI.
23. Russell DW. 2003. The enzymes, regulation, and genetics of bile acid synthesis. Annual
Review of Biochemistry 72: 137–74.
24. Hagey LR, Molller PR, Hofmann AF, Krasowski MD. 2010. Diversity of bile salts in fish
and amphibians: evolution of a complex biochemical pathway. Physiological and
Biochemical Zoology: PBZ 83: 308–21.
25. Siefkes MJ, Li W. 2004. Electrophysiological evidence for detection and discrimination of
pheromonal bile acids by the olfactory epithelium of female sea lampreys (Petromyzon
marinus). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and
Behavioral Physiology 190: 193–9.
28

26. Hofmann AF, Mysels KJ. 1987. Bile salts as biological surfactants. Colloids and Surfaces
30: 145–73.
27. Houten SM, Watanabe M, Auwerx J. 2006. Endocrine functions of bile acids. The EMBO
Journal 25: 1419–25.
28. Kittredge JS, Takahashi FT. 1972. The evolution of sex pheromone communication in the
Arthropoda* 1. Journal of Theoretical Biology 35: 467–71.
29. Polkinghorne CN, Olson JM, Gallaher DG, 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 Physiology and Biochemistry 24: 15–30.
30. Zhang C, Brown SB, Hara TJ. 1996 Olfactory sensitivity and reactions of lake char to bile
acids released by conspecifics. Chemical Senses 21:692–93
31. Sato K, Suzuki N. 2001. Whole-cell response characteristics of ciliated and microvillous
olfactory receptor neurons to amino acids, pheromone candidates and urine in rainbow
trout. Chemical Senses 26: 1145 –56.
32. Hubbard P, Barata E, Canário A. 2003. Olfactory sensitivity of the gilthead seabream
(Sparus auratus L.) to conspecific body fluids. Journal of Chemical Ecology 29: 2481–
98.
33. Huertas M, Hubbard PC, Canário AVM, Cerdà J. 2007. Olfactory sensitivity to
conspecific bile fluid and skin mucus in the European eel Anguilla anguilla (L.). Journal
of Fish Biology 70: 1907–20.
34. Stabell OB, Selset R. 1980. Comparison of mucus collecting methods in fish olfaction. Acta
Physiologica Scandinavica 108: 91–6.
35. Saglio P, Fauconneau B. 1985. Free amino acid content in the skin mucus of goldfish,
Carassius auratus L.: influence of feeding. Comparative Biochemistry and Physiology 82:
67–70.
36. Vermeirssen E, Scott A. 2001. Male priming pheromone is present in bile, as well as urine,
of female rainbow trout. Journal of Fish Biology 58: 1039–45.
37. Appelt CW, Sorensen PW. 2007. Female goldfish signal spawning readiness by altering
when and where they release a urinary pheromone. Animal Behaviour 74: 1329–38.
38. Kawabata K. 1993. Induction of sexual behavior in male fish (Rhodeus ocellatus ocellatus)
by amino acids. Amino Acids 5: 323–7.
39. Scott A, Canario A, Sherwood NM, Warby CM. 1991. Levels of steroids, including
cortisol and 17α20βdihydroxy-4-pregnen-3-one, in plasma, seminal fluid, and urine of
29

Pacific herring (Clupea hareng us pallasi) and North Sea plaice (Pleuronectes platessa
L.). Canadian Journal of Zoology 69: 111–6.
40. Resink J, Schoonen W, Albers P, File D, et al. 1989. The chemical nature of sex attracting
pheromones from the seminal vesicle of the African catfish, Clarias gariepinus.
Aquaculture 83: 137–51.
41. De Witt EH, Lack L. 1980. Effects of sulfation patterns on intestinal transport of bile salt
sulfate esters. American Journal of Physiology: Gastrointestinal and Liver Physiology
238: G34–39.
42. Venkatachalam K. 2005. Petromyzonol sulfate and its derivatives: the chemoattractants of
the sea lamprey. BioEssays 27: 222–8.
43. Johnson NS, Yun S‐S, Buchinger TJ, Li W. 2012. Multiple functions of a multi‐component
mating pheromone in sea lamprey Petromyzon marinus. Journal of Fish Biology 80: 538–
54.
44. Fine J, Sorensen P. 2010. Production and fate of the sea lamprey migratory pheromone.
Fish Physiology and Biochemistry 36: 1013–20.
45. Shimada K, Bricknell KS, Finegold SM. 1969. Deconjugation of bile acids by intestinal
bacteria: review of literature and additional studies. The Journal of Infectious Diseases :
119: 273–81.
46. Ternes TA, Kreckel P, Mueller J. 1999. Behaviour and occurrence of estrogens in
municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge.
The Science of the Total Environment. 225: 91–9.
47. Aries V, Hill MJ. 1970. Degradation of steroids by intestinal bacteria II. Enzymes catalysing
the oxidoreduction of the 3 [alpha]-, 7 [alpha]-and 12 [alpha]-hydroxyl groups in cholic
acid, and the dehydroxylation of the 7-hydroxyl group. Biochimica et Biophysica Acta:Lipids and Lipid Metabolism. 202: 535–43.
48. Xi X, Johnson NS, Brant CO, Yun SS, et al. 2011. Quantification of a Male Sea Lamprey
Pheromone in Tributaries of Laurentian Great Lakes by Liquid Chromatography Tandem
Mass Spectrometry. Environmental Science & Technology. 45: 6437–43.
49. Sorensen PW, Scott AP, Kihslinger RL. 2000. How common hormonal metabolites
function as relatively specific pheromonal signals in the goldfish. In: International
Symposium on the Reproductive Physiology of Fish. p 125–8.
50. Eschmeyer PH. 1956. The early life history of the lake trout in Lake Superior. Michigan
Department of Conservation, Institute of Fisheries Research. Miscellaneous Publication
10: 1–31.

30

51. Royce WF. 1951. Breeding habits of lake trout in New York. U.S. Fish and Wildlife Service
Fishery Bulletin. 52: 59–76.
52. Foster NR. 1985. Lake trout reproductive behavior: influence of chemosensory cues from
young-of-the-year by-products. Transactions of the American Fisheries Society 114:
794–803.
53. Stabell OB. 1987. Intraspecific pheromone discrimination and substrate marking by Atlantic
salmon parr. Journal of Chemical Ecology 13: 1625–43.
54. Hara TJ. 1994. The diversity of chemical stimulation in fish olfaction and gustation.
Reviews in Fish Biology and Fisheries 4: 1–35.
55. Erickson JR, Caprio J. 1984. The spatial distribution of ciliated and microvillous olfactory
receptor neurons in the channel catfish is not matched by a differential specificity to
amino acid and bile salt stimuli. Chemical Senses 9: 127 –41.
56. Huertas M, Hagey L, Hofmann A, Cerdà J, et al. 2010. Olfactory sensitivity to bile fluid
and bile salts in the European eel (Anguilla anguilla), goldfish (Carassius auratus) and
Mozambique tilapia (Oreochromis mossambicus) suggests a “broad range” sensitivity not
confined to those produced by conspecifics alone. Journal of Experimental Biology 213:
308–17.
57. Sorensen PW, Hara TJ, Stacey NE. 1987. Extreme olfactory sensitivity of mature and
gonadally-regressed goldfish to a potent steroidal pheromone, 17$\alpha$, 20$\beta$dihydroxy-4-pregnen-3-one. Journal of Comparative Physiology A: Neuroethology,
Sensory, Neural, and Behavioral Physiology 160: 305–13.
58. Sorensen PW. 1986. Origins of the freshwater attractant (s) of migrating elvers of the
American eel, Anguilla rostrata. Environmental Biology of Fishes 17: 185–200.
59. Ferrari MCO, Wisenden BD, Chivers DP. 2010. Chemical ecology of predator-prey
interactions in aquatic ecosystems: a review and prospectus The present review is one in
the special series of reviews on animal-plant interactions. Canadian Journal of Zoology
88: 698–724.
60. Mathis A, Smith RJF. 1993. Fathead minnows, Pimephales promelas, learn to recognize
northern pike, Esox lucius, as predators on the basis of chemical stimuli from minnows in
the pike’s diet. Animal Behaviour 46: 645–56.
61. Dillon RJ, Vennard CT, Charnley AK, et al. 2000. Exploitation of gut bacteria in the
locust. Nature 403: 851.
62. Jones K, Hara T. 1985. Behavioural responses of fishes to chemical cues: results from a
new bioassay. Journal of Fish Biology 27: 495–504.

31

63. Nordeng H. 1977. A pheromone hypothesis for homeward migration in anadromous
salmonids. Oikos 28: 155–9.
64. Shoji T, Ueda H, Ohgami T, Sakamoto T, et al. 2000. Amino acids dissolved in stream
water as possible home stream odorants for masu salmon. Chemical Senses 25: 533–40.
65. Shoji T, Yamamoto Y, Nishikawa D, Kurihara K, et al. 2003. Amino acids in stream
water are essential for salmon homing migration. Fish Physiology and Biochemistry 28:
249–51.
66. Hasler AD, Wisby WJ. 1951. Discrimination of stream odors by fishes and its relation to
parent stream behavior. American Naturalist : 223–38.
67. Robinson TC, Sorensen PW, Bayer JM, Seelye JG. 2009. Olfactory sensitivity of Pacific
lampreys to lamprey bile acids. Transactions of the American Fisheries Society 138:
144–52.
68. Selset R, Døving KB. 1980. Behaviour of mature anadromous char (Salmo alpinus L.)
towards odorants produced by smolts of their own population. Acta Physiologica
Scandinavica 108: 113–22.
69. Ueda H, Yamamoto Y, Hino H. 2007. Physiological mechanisms of homing ability in
sockeye salmon: from behavior to molecules using a lacustrine model. American
Fisheries Society Symposium 54: 5–16.
70. Groot C, Quinn T, Hara T. 1986. Responses of migrating adult sockeye salmon
(Oncorhynchus nerka) to population-specific odours. Canadian Journal of Zoology 64:
926–32.
71. Yamamoto Y, Hino H, Ueda H. 2010. Olfactory imprinting of amino acids in lacustrine
sockeye salmon. PloS one 5: e8633.
72. Yambe H, Kitamura S, Kamio M, Yamada M, et al. 2006. L-Kynurenine, an amino acid
identified as a sex pheromone in the urine of ovulated female masu salmon. Proceedings
of the National Academy of Sciences 103: 15370–4
73. Sveinsson T, Hara TJ. 1995. Mature males of Arctic charr, Salvelinus alpinus, release Ftype prostaglandins to attract conspecific mature females and stimulate their spawning
behaviour. Environmental Biology of Fishes 42: 253–66.
74. Huertas M, Scott AP, Hubbard PC, Canário AVM, et al. 2006. Sexually mature European
eels (Anguilla anguilla L.) stimulate gonadal development of neighbouring males:
possible involvement of chemical communication. General and Comparative
Endocrinology 147: 304–13.

32

75. Johnson NS, Yun SS, Thompson HT, Brant CO, et al. 2009. A synthesized pheromone
induces upstream movement in female sea lamprey and summons them into traps.
Proceedings of the National Academy of Sciences 106: 1021–6.

33

CHAPTER 2

ORIGIN OF A BILE ALCOHOL MATING SIGNAL IN AN ANCIENT AGNATHAN

34

ABSTRACT

The diversity of sexual signals is perplexing, with many signals influencing decisions for
which they possess little inherent information. The sensory bias hypothesis suggests that female
preference for seemingly unmeaningful signals evolves in a non-sexual context, and that males
manipulate the pre-existing preference by signaling with a matching stimulus. I postulated that
the male-released bile alcohol mating pheromone, 3 keto petromyzonol sulfate (3kPZS), of sea
lamprey (Petromyzon marinus) evolved as a result of a sensory bias. I evaluated the hypothesis
by comparing the behavior and physiology of sea lamprey to that of the ancestral silver lamprey
(Ichthyomyzon unicuspis). Larval silver lamprey holding waters contained high concentrations of
3kPZS. In laboratory and simulated stream environments, both migratory and spawning-phase
female silver lamprey were more likely to move upstream when 3kPZS was applied, but were
not attracted to the source. Male silver lamprey did not biosynthesize or release 3kPZS at rates
sufficient to be detected by females in natural high-volume stream environments. I infer that
female silver lamprey evolved to cue onto 3kPZS excreted by stream-resident larvae as a
mechanism to locate habitat conducive to offspring growth and survival, and that males do not
manipulate the female preference by signaling with 3kPZS.

35

INTRODUCTION

Biologists have long been fascinated with natural complements, specifically concerning
the evolution of matching pairs (i.e. receptor and ligand) [1]. Such questions are particularly
interesting in understanding the origins of sexual communication systems, where the pair of
interest is often female preference and male signal. In some circumstances, the origin of
preference/signal pairs has been explained by sensory biases, where males manipulate female
behavior with a signal which overlaps with the female’s pre-existing preferences [2]. Contrary to
Fisherian and indicator trait models which suggest co-evolution of preference and signal, sensory
bias suggests that female preference pre-dates male signaling [3]. The primary facet of sensory
bias as a mechanism for the origin of sexual communication is the molding of the female
preference via natural selection, rather than sexual selection. Empirical discussions regarding
sensory biases developing into sexual communication include that of the guppy (Poecilia
reticulate), where the nuptial colors of male guppies match female preferences for orange fruits
[4], and the swordtail fishes (Xiphophorus sp.), where female preference predates male
possession of swords [5]. Such examples may not conclusively support sensory bias over
alternative models as the female preference, although predating male signal, may have evolved
via sexual selection rather than natural selection [6].
Further contributing to gaps in understanding origins of female preferences is the focus of
research on the auditory and visual sensory modalities. Sexual signaling via chemoreception,
likely the most primitive and ubiquitously used sense [7, 8], is rarely considered in
communication theory [9]. The identification of male insect pheromones as floral compounds
has been followed by hypotheses of the involvement of sensory bias in the evolution of female

36

preferences [7], but further empirical evidence is sparse. Research into chemical communication
between fish has resulted in similar hypotheses of the origin of odor preferences [10], where sex
steroids [11], prostaglandins [12], amino acids [13], and bile acids [14] are detected with acute
sensitivity and specificity, and influence receiver behavior. Although it has been suggested that
preference for the above compounds may have evolved under natural selection, experimental
evidence has yet to be provided. A promising model system for investigating the origins of
chemical communication between fish is that of the sea lamprey (Petromyzon marinus).
Sea lamprey rely upon olfaction to complete the final stages of their semelparous life
[15]. Conspecific odors facilitating the migration of reproductive adults, and subsequent act of
spawning, have been of particular interest [16, 17]. Bile acids emanating from stream-resident
larvae direct adults from lakes or the ocean into streams with a recent history of high offspring
survival [17]. Upon sexual maturation, males release large amounts of 7α, 12α, 24-trihydroxy-5α
-cholan-3-one-24-sulfate (3 keto petromyzonol sulfate, 3kPZS) via glandular cells in the gills
[14, 18], which is a potent attractant of sexually mature females [19, 20]. Although the
biosynthetic pathway of 3kPZS is yet to be elucidated, one hypothesis is that similar
petromyzontid bile acids are intermediates in the hepatic biosynthetic pathway, ending with
3α,7α,12α,24-tetrahydroxy-5α-cholan-24-sulfate (petromyzonol sulfate, PZS) which is then
transported from the liver to the gills and oxidized, forming 3kPZS [21].
Sensory bias as a mechanism of signal evolution has been considered when two lines of
evidence are present: 1) the shaping of female preference by natural selection, rather than sexual
selection, and 2) female preference and lack of male signaling in closely related, but more
primitive species [22, 23]. I use a similar approach to provide empirical evidence for sensory
bias as the origin for female preference by comparing the behavior and physiology of the sea

37

lamprey and the silver lamprey (Ichthyomyzon unicuspis), which is thought to be the ancestral
species of the Petromyzontidae family [24, 25]. I hypothesized that 1) the female preference for
3kPZS evolved in the silver lamprey under natural selection, functioning to direct migratory
adults into habitat conducive to high offspring growth and survival, and 2) male silver lamprey
have not manipulated female preference by releasing large amounts of 3kPZS. According to
these hypotheses, I predicted that a) larval silver lamprey excrete 3kPZS into the water, b)
migratory and spawning female silver lamprey are more likely to move upstream when 3kPZS is
present, and c) male silver lamprey do not release large amounts of 3kPZS, or similar bile acid
derivatives, into the water.

38

METHODS
Experimental animals
Experimental fish were used with approval from Michigan State University’s Animal Use
and Care Committee (Approval # 04/10-043-00). Larvae were collected via backpack
electroshocking and adults were caught in United States Fish and Wildlife Service sea lamprey
traps. Larvae were immediately used in experiments, while adult lamprey were transported to
Hammond Bay Biological Station, Millersburg, MI and stored in 4 °C 1000 L flow through
tanks. Females were distinguished from males based on a large, soft abdomen. Ovulation or
spermiation was determined based on the expression of eggs or milt with gentle manual pressure
[18]. All sample collection and behavioral experiments were conducted between sunset and
sunrise due to the nocturnal behavior of lampreys.

Release of bile acids

Holding water and tissue collections
Bile acid release by larvae was investigated following methods similar to those used in
the collection of sea lamprey larval odors [26]. However, release experiments were conducted
immediately following collection, due to concerns of potential bile acid release changes resulting
from artificial holding and feeding. Silver lamprey and northern brook lamprey (I. fossor) are
indistinguishable as larvae, and may be two morphs of the same species [27]. To determine if
larvae of each species release different amounts of 3kPZS, larvae were collected from a stream
segment thought to contain mostly silver lamprey and a second stream segment thought to
contain mostly northern brook lamprey. Thirty larvae (weight = 2.33 ± 0.12 g, length = 109.4 ±

39

1.71 mm; mean ± se) were sampled from each collection site across the reproductive season of
lamprey (May-July, N=7). After collection, larvae were divided into three groups of 10 and
placed in a 5 L container with 5 cm of sand, aeration, and supplied with ambient Lake Huron
water. Following a 24 hr acclimation period, the inflow of water was shut off, the lake-water
decanted, and 4 L deionized water added. The odor of larvae was allowed to accumulate for 12
hr overnight, after which a 1 L water sample was spiked with a 5-deutarated 3kPZS (5-d 3kPZS)
[28] internal standard and stored at less than -20 °C until bile acids were quantified. Previous
experiments found minimal 3kPZS in Lake Huron water void of larvae (< 0.1 ng/ml). The
influence of species and date on the release of 3kPZS was evaluated using an ANOVA.
Bile acid production and release by adults was investigated by collecting holding waters
and tissues from preovulated (weight = 47.2 ± 0.64 g, length = 275.81 ± 1.51 mm; mean ± se)
and ovulated (weight = 58.22 ± 1.56 g, length =277.1 ± 2.23 mm; mean ± se) female, and
prespermiating (weight =43.83 ± 0.58 g, length = 281.06 ± 1.33 mm; mean ± se) and spermiating
(weight =40.25 ± 0.37 g, length = 258.83 ± 0.76 mm; mean ± se) male silver lamprey.
Spermiated male sea lamprey (weight = 236.75 ±4.44 g, length = 436.33 ± 15.98 mm; mean ±
se) were also sampled to provide a point of reference. A single individual was held in 5 L of
aerated, deionized water. After 2 hr, a 250 ml water sample was spiked with 5-d 3kPZS and
stored at less than -20 °C for later bile acids quantification. Lamprey were then euthanized with
an overdose of 3-aminobenzoic acid ethyl ester (MS-222; www.sigmaaldrich.com) and
dissected. Liver tissues (bile acid biosynthesis) and gill tissues (bile acid release) were either
fixed in 4% paraformaldehyde in 0.1M phosphate buffer for immunocytochemistry or frozen at 80 °C for bile acid extraction. The influence sex, maturity, and species on bile acid concentration
was evaluated using an ANOVA and a post-hoc Tukeys test (α = 0.05).

40

Bile acid quantification
Bile acids in tissue samples were extracted following established methods [29]. Briefly,
5d-3kPZS internal standard was added to each sample, tissues were homogenized in 75%
ethanol, and centrifuged at 13,000 rpm at room temperature for 5 minutes. One ml of -20°C
acetonitrile was added to the supernatant to precipitate any proteins. The mixture was centrifuged
at 13,000 at 4 °C for 10 minutes. Finally, the supernatant was evaporated, and reconstituted in
50% methanol/H20. A 10 ml subsample from each water sample was evaporated using a
CentriVap Cold Trap with CentriVap Concentrator (Labconco Co. MO, USA,
www.labconco.com) and reconstituted in 50% methanol. Concentrated bile acids were subjected
to ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLCMS/MS) following methods described by Li et al. [28]. Treatments that had less than two
replicates with mass-spectrometer signal to noise ratio more than 10 were not included in the
analyses. Eight bile acids putatively involved in the reproductive behavior of lamprey were
quantified (Table 1).

41

Table 1. Bile salts putatively involved in reproductive behavior of the sea lamprey (Petromyzon
marinus).
Abbreviation
ACA
PZ
PZS
3kPZS
3kACA
3kPZ
PADS

PSDS

Common name
allocholic acid
petromyzonol
petromyzonol sulfate
3keto petromyzonol
sulfate
3keto allocholic acid
3keto petromyzonol
petromyzonamine
disulfate
Petromyzonsterol
disulfate

Chemical name
3α,7α,12α-Trihydroxy-5α-cholan-24-oic-acid
3α,7α,12α,24-tetrahydroxy-5α-cholane
3α,7α,12α,24-Tetrahydroxy-5α-cholan-24-sulfate
7α, 12α, 24-trihydroxy-5α -cholan-3-one-24sulfate
7 α,12α -dihydroxy-5α -cholan-3-one-24-oic acid
7α,12α,24-trihydroxy-5α-cholane-3-one
(3β, 5α,7α, 24R)-1-[3-[[7,24bis(sulfooxy)cholestan-3-yl]amino]-propyl]-2pyrrolidinone, disodium salt
12-hydroxy-5, 22-diene-ergosta-3,28-disulfate

42

Immunocytochemistry
The Michigan State University Histopathology Lab mounted gill and liver tissues in
paraffin, and stained one set of slides with hemotoxylin and eosin (H&E). For
immunocytochemistry, unstained gill and liver tissues were deparaffinized with three 5 min
washes in xylene, and rehydrated using serial ethanol washes from 100% to 10%. Tissues were
rinsed with Tris buffer saline (TBS; 50 mM Tris, 150 mM NaCl, pH 7.2) between each of the
following treatments. Endogenous peroxidase activity was eliminated with a 10 min 0.01% H2O2
(DAB substrate kit, Vector) treatment. Tissues were then incubated at 4 °C for 24 hr with the
primary 3kPZS antibody (1:1000, Rabbit IgG; GenScript USA Inc., http://www.genscript.com
and normal goat serum diluted in 50 mM TBS plus 0.05% Triton X-100). The anti-body used
does not discriminate between 3kPZS, 3kACA, or 3kPZ [18]. A second set of tissues consisting
of one slide per experimental group was incubated with normal goat serum as negative controls,
which showed no immunopositive staining. The primary antibody was amplified using a 2 hr
treatment with Alexafluor 594 goat anti-rabbet IgG. The tissues were mounted with ProLong
Gold antifade reagent with DAPI (Life Technologies Co., www.lifetechnologies.com).

Behavioral response to 3kPZS

Two-choice maze
The two-choice maze design and method used by Li et al. [14] was used to determine the
-12

near-source preference of preovulated and ovulated silver lamprey to 1x10

M 3kPZS (Figure

2-1A). The concentration of 3kPZS used matched that of previous studies [20]. Briefly, a single
lamprey was introduced to the furthest downstream point of the maze. After a ten minute
acclimation to the maze, the time the animal spent in each channel was recorded by hand and by
43

an infrared camera. After twenty minutes of recording, 3kPZS dissolved in 50% methanol was
introduced to a random side, along with a 50% methanol control to the opposing side. The odor
was pumped into the maze for 5 minutes without recording the lamprey’s behavior. After 5
minutes, the behavior was recorded for another twenty minutes. After recording the time spent
in the control and experimental channels prior to odor application (BC, BE), and the time spent
in the control and experimental channels after odor application (AC, AE), an index of preference
was calculated for each test (I = Ae/Be - Ac/Bc). The indices of preference were evaluated using
a Wilcoxon Signed-rank test (α= 0.05) [14].

Quasi-natural stream
The response of preovulated and ovulated female silver lamprey, and ovulated sea
-12

lamprey to 1x10

M 3kPZS was also evaluated in a quasi-natural stream system (Figure 2-1B).

Four passive integrated transponder (PIT) antennae were constructed to monitor the movement
of lamprey. One upstream of the release cage, one downstream of the release cage, and one at
each of two nests constructed at the uppermost point of the system. Prior to each trial, 2-5
lamprey we implanted with 12 mm PIT tags (Oregon RFID, Portland, OR) and placed in an
acclimation cage in the stream adjacent to the constructed system, at least 18 hr (migratory trials)
or 8 hr (spawning-phase trials) prior to experimentation. Thirty minutes prior to experimentation,
lamprey were moved from the acclimation cage to the release cage in the experimental system.
The experiment began with the application of an odor into the assigned channel. Fifteen minutes
after odor application, lamprey were released from the acclimation cage and allowed to swim
freely for 1 hr. Movement of lamprey upstream of the release cage and the first choice nest
selection based upon odor application were evaluated as binary data using logistic regression
44

-12

[20]. Two treatments were applied to the channels; 1) 1 x 10

M 3kPZS vs. Methanol, and 2)

Methanol vs. Methanol. The treatments were alternated and lamprey were only used once for
each treatment. Logistic regression models showed no evidence of over dispersion.

45

B

A

10 m

Release

1m

1m

Figure 2-1. Assays used to evaluate the behavioral responses of silver lamprey to 3kPZS. A)
Two-choice maze modified from Li et al. [2002] used to evaluate the proximate response of
silver lamprey to 3kPZS. The size was scaled down to half the size of that used for sea lamprey,
as silver lamprey are approximately half the size of sea lamprey. B) Quasi-natural stream used
to evaluate response of silver lamprey to 3kPZS in a natural setting. Large dashed lines represent
overhead-flow boards used to reduce turbulence. Small dashed lines represent mesh restricting
movement of lamprey. Arrows represent direction of flow. Dark bars represent PIT antennae
used to monitor movement of lamprey. Double angled-dashes represent breaks in the scale of the
large stream-like system, allowing a side-by-side comparison for the purposes of the figure.
46

RESULTS

Bile acid excretion by larvae
Release rates of 3kPZS, PZS, and PADS by larval silver lamprey were 40.75 ± 8.8, 4.76
± 1.85, and 1.1 ± 0.36 ng/larva/hr, respectively (mean ± se; Figure 2-2). All other compounds
were released at rates less than 1 ng/larva/hr. Concentrations of 3kPZS in the water did not
change across the months sampled (F3, 17 =1.21, p=0.3377), indicating that the release rate is
similar across the entire reproductive season of lamprey. The release of 3kPZS was not
significantly different between larvae from the two stream-segments (F1, 19 =0.528, p=0.4763),
indicating no difference in 3kPZS release between silver lamprey and northern brook lamprey.
Accordingly, release rates reported were the mean of all samples combined.

47

50
45
40

ng/larva/ hr

35
30
25
20
15
10
5
0

3kPZS

PZS

PADS

Figure 2-2. Mean rate (ng/larva/hr ± standard error) at which silver lamprey larvae released 3
keto petromyzonol sulfate (3kPZS), petromyzonol sulfate (PZS), and petromyzonamine disulfate
(PADS) into the water.

48

Bile acid biosynthesis and release by adults
Immunocytochemistry and UPLC-MS/MS experiments conducted to investigate the
hepatic biosynthesis of Petromyzontid bile acids indicated that male silver lamprey liver tissue
contains low concentrations of 3kPZS relative to sea lamprey. Immunopositive staining was
strong but sparsely scattered in the hepatocyte cytoplasm and cytoplasmic granule of both
prespermiating and spermiating male silver lamprey (Figure 2-3). Staining was absent in
preovulated and ovulated females. UPLC-MS/MS analysis of silver lamprey liver tissue detected
only 3kPZS, PZS, and PADS at concentrations greater than 1 ng/g tissue (Figure 2-4). The
concentrations of 3kPZS, PZS, and PADS were highest in spermiated sea lamprey and did not
differ between sex or maturity combination of silver lamprey (F 4, 20 = 8.46, p < 0.001, F 4, 20 =
47.76, p < 0.001, F 4, 17 = 8.83, p < 0.001, respectively, post hoc Tukey’s test α = 0.05).

49

A

B

Figure 2-3. Immunopossitive staining (orange) to the 3 keto antibody in the liver tissue of
spermiated (A) and prespermiated (B) male silver lamprey. Scale-bar is 20µm. Both images
were taken using an X40 objective and the same exposure settings. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this thesis.

50

10000
1000

ng/g tissue

100

10
1

0.1
0.01
0.001

IFSiL

MFSiL

IMSiL

MMSiL

MMSL

Figure 2-4. Mean concentration of 3 keto petromyzonol sulfate (3kPZS; black), petromyzonol
sulfate (PZS; dark grey) and petromyzonamine disulfate (PADS; light grey; ng/g tissue ±
standard error) detected in the liver tissues of silver and sea lamprey, presented on a log scale. IF
Sil = immature female silver lamprey, MF SiL = mature female silver lamprey, IM SiL =
immature male silver lamprey, MM SiL = mature male silver lamprey, and MM SL = mature
male sea lamprey.

51

Interplatelet regions of the spermiated male silver lamprey gill filaments contained large
columnar glandular cells possessing a basally located nucleus (Figure 2-5A). Gill tissues of
prespermiated males did not possess glandular cells (Figure 2-5B). Gill tissues of spermiated
males showed scattered weak staining localized to platelet cells and interplatelet glandular cells
(Figure 2-5C). Immunopositive staining in prespermiated males was scattered and weak, but
localized to platelet cells (Figure 2-5D).
UPLC-MS/MS analysis of silver lamprey gill tissues detected only 3kPZS and PZS at
concentrations greater than 1 ng/g tissue (Figure 2-6). The concentrations of 3kPZS and PZS
were highest in spermiated sea lamprey and did not differ between sex or maturity combination
of silver lamprey (F 3, 20 = 18.39, p < 0.001, F 3, 20 = 45.59, p < 0.001, respectively, post hoc
Tukey’s test α = 0.05). Concentrations of bile acids in ovulated female gills were not determined
due to lack of animals.
UPLC-MS/MS analysis of silver lamprey holding waters detected only 3kPZS
concentrations greater than 1 ng/g individual/hr (Figure 2-7). The concentration of 3kPZS was
highest in spermiated sea lamprey and did not differ between sex or maturity combination of
silver lamprey (F 4, 68 = 179.35, p < 0.001, post hoc Tukey’s test α = 0.05). One outlier was
removed from spermiated silver lamprey (1009.16 ng 3kPZS/g lamprey/hr) as it was
approximately five standard deviations from the mean.

52

A

B

C

D

Figure 2-5. Histology of male silver lamprey gill tissues. H&E stained gill tissues of spermiating
males (A) show cells similar to previously documented glandular cells (large arrows). H&E
stained gill tissues of prespermiated males (B) do not show glandular cells. Immunoreactive
3kPZS (orange) in the gill tissue of spermiating (C) males was weak but localized in platelet
(small arrow) and glandular (large arrow) cells and in platelet cells (small arrow) of
prespermiating (D) males. Scale-bar is 20µm. Both images were taken using an X40 objective
and the exposure settings.

53

1000

100

ng/g tissue

10

1

0.1

0.01

NA

0.001

IFSiL

MFSiL

IMSiL

MMSiL

MMSL

Figure 2-6. Mean concentration of 3 keto petromyzonol sulfate (3kPZS; black) and
petromyzonol sulfate (PZS; dark grey; ng/g tissue ± standard error) detected in the gill tissues of
silver lamprey, presented on a log scale. IF Sil = immature female silver lamprey, MF SiL =
mature female silver lamprey, IM SiL = immature male silver lamprey, MM SiL = mature male
silver lamprey, and MM SL = mature male sea lamprey. NA = samples not able to be taken.

54

1000

ng/g individual/hr

100

10

1

0.1

0.01

0.001

IFSiL

MFSiL

IMSiL

MMSiL

MMSL

Figure 2-7. Mean release (ng/g individual/hr) of 3 keto petromyzonol sulfate (3kPZS) into the
water as quantified with UPLC-MS/MS, presented on a log scale. IF Sil = immature female
silver lamprey, MF SiL = mature female silver lamprey, IM SiL = immature male silver lamprey,
MM SiL = mature male silver lamprey, and MM SL = mature male sea lamprey.

55

Behavioral response of adult female silver lamprey to 3kPZS
Preovulated and ovulated female silver lamprey showed no preference for 1x 10

-12

M

synthesized 3kPZS over methanol in two-choice maze experiments (P > 0.05, Figure 2-8).
Results from the constructed stream system confirmed those of the two-choice maze; the entry of
preovulated and ovulated female silver lamprey into 3kPZS-baited nests was not significantly
different from that into methanol-baited nests (χ2= 0.68, df = 1, P = 0.41, χ2 = 1.36, df = 1, P =
0.24, respectively, Figure 2-9). Sea lamprey were significantly lured to the source of 3kPZS (χ2=
14.42, df = 1, P = <0.001). The upstream movement of both preovulated and ovulated female
silver lamprey was influenced by 3kPZS, with a significantly higher proportion of females
moving upstream of the release cage when 3kPZS was applied than when methanol was applied
(χ2= 4.62, df = 1, P = 0.032, χ2 = 21.38, df = 1, P = <0.001, respectively, Figure 2-10).

56

1
0.8
0.6

*

Preference

0.4
0.2
0
-0.2

IFSiL

MFSiL

MFSL

-0.4
-0.6
-0.8
-1

Figure 2-8. Near-source preference of silver lamprey of each sex and maturity combination to 3
keto petromyzonol sulfate (3kPZS) as evaluated using a two-choice maze. Error bars represent
the standard error of the mean. The response of ovulated female sea lamprey to 3kPZS is shown
for reference (N. Johnson, unpublished data). The asterisk represents significance as determined
using a Wilcoxon signed rank test. IF Sil = immature female silver lamprey, MF SiL = mature
female silver lamprey, and MF SL = mature female sea lamprey.

57

*

1
0.9
0.8

Enter odor

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

4/6

2/6

IFSiL

8/12

4/12

MFSiL

0/11

11/11

MFSL

Figure 2-9. Near-source behavior of migratory and spawning female silver lamprey and
spawning sea lamprey in the quasi-natural stream experiments. Data are presented as the mean
proportion of females that entered the 3 keto petromyzonol sulfate (3kPZS) baited nest (dark
grey) or the methanol baited nest (light grey). Error bars represent the standard error of the mean.
Significant differences between the response to 3kPZS and response to methanol (α = 0.05) as
determined using logistic regression are displayed with an asterisk. N/N = the number of lamprey
that enter the odor/ total number of lamprey that made the choice between odors. IF Sil =
immature female silver lamprey, MF SiL = mature female silver lamprey, and MF SL = mature
female sea lamprey.

58

*

1
0.9

Proportion upstream

0.8

0.7
0.6
0.5

*
0.4
0.3
0.2
0.1
0

11/30

3/25

14/15

IFSiL

1/13

MFSiL

11/20

8/15

MFSL

Figure 2-10. Upstream movement of migratory and spawning female silver lamprey and
spawning sea lamprey in quasi-natural stream experiments. Data are presented as the mean
proportion of females that moved upstream when 3 keto petromyzonol sulfate (3kPZS; dark
grey) or methanol (light grey) was applied. Error bars represent the standard error of the mean.
Significant differences (α = 0.05) between the response to 3kPZS and the response to methanol
as determined using logistic regression are displayed with an asterisk. N = the number of
lamprey that move upstream/total number of lamprey released. IF Sil = immature female silver
lamprey, MF SiL = mature female silver lamprey, and MF SL = mature female sea lamprey.

59

DISCUSSION

Sensory bias models explaining the evolution of female preferences and male signals
have been evaluated by providing evidence for 1) the shaping of female preference under natural
selection and 2) the lack of male signaling in an ancestral species [22, 23]. Here, I studied the
chemical communication system of lamprey, and provide evidence for the influence of sensory
biases on sexual signaling. Spermiated male sea lamprey direct ovulated females to nests by
releasing a bile alcohol signal, 3kPZS, at high rates via glandular cells in the gills [14, 18, 19,
20]. Bile alcohols likely hold very little inherent information as to the reproductive fitness of an
individual (Chapter 1), thus I postulated that the female preference for 3kPZS evolved through a
non-sexual mechanism. Specifically, I hypothesized that the ancestral silver lamprey cue onto
larval-released 3kPZS to locate streams containing habitat conducive to offspring survival and
that males have not adapted to manipulate such preference by signaling with 3kPZS. According
to our predictions, (a) 3kPZS was found in high concentration in larval holding waters, (b) more
migratory females move upstream in response to 3kPZS, and spawning-phase females retain the
upstream movement response to 3kPZS, and (c) spawning-phase males do not biosynthesize or
release 3kPZS or similar bile acids at rates sufficient to be detected by females. Based upon the
above findings, I infer that the female preference for 3kPZS evolved via natural selection in
silver lamprey, but male signaling evolved later in the sea lamprey.
The contrasting mating strategies employed by sea lamprey and silver lamprey likely
contribute to the differences in chemical signaling between the two species. Sea lamprey are
polygynous, with intense male competition and aggression. Silver lamprey, however, are
polygynandrous, with communal spawning groups of males and females [30]. The selective
pressure of a polgynous male to attract females to the spawning nest will be much stronger than
60

that of a polgynadrous male. The weaker selective pressure on silver lamprey males to signal
females could be suggested as alternative interpretation of the data. For example, individual
males may not need to synthesize and release large amounts of 3kPZS into the water to attract
females, as they share the burden of luring females with multiple males. However, this
alternative does not seem feasible, given the low rate at which male silver lamprey release
3kPZS (~200 ng/male/hr). Electrophysiological studies indicate that female sea lamprey are
12

unable to detect 3kPZS at concentrations below 1x 10-

M [31]. Activating the quasi-natural

stream used in the present study, which had water flow similar or less than natural silver lamprey
spawning habitat, would require a nest of over 200 male silver lamprey. Reported silver lamprey
nests were occupied by no more than 15 individuals [32]. Furthermore, the inability of ovulated
female silver lamprey to locate the source of 3kPZS follows the hypothesis that the female
preference evolved as a migratory behavior via natural selection, and is not a response to the
minute amount of 3kPZS released by males. Larval habitat and spawning habitat do not overlap,
as larvae require mucky sediment in which to burrow [33], and spawners require rocky substrate
in which to construct nests [30]. Thus, although entering the stream containing larval odor is
likely adaptive for females, locating the source of larval odor is unlikely adaptive for females
given males have not evolved to signal with 3kPZS.
The rates of synthesis and release indicate the biological function of 3kPZS and male
glandular cells may be related to the physiology of the male rather than sexual communication
between individuals. Spermiating male silver lamprey possessed minute but quantifiable
concentrations of 3kPZS in tissue and water samples, and cells similar to the glandular cells
previously suggested to release 3kPZS in sea lamprey [18]. One hypothesis could be that the
glandular cells play a role in osmoregulation, and that the mitochondria-rich cells represent ion61

regulating chloride cells [34, 35]. However, this hypothesis can be discounted as females face the
same osmoregulatory challenges, but fail to develop glandular cells. A second hypothesis could
be that lampreys use glandular cells to eliminate bile acids of the body after the bile duct
atrophies [36]. However, if bile acid elimination were the function one would expect that 1) the
glandular cells would develop much earlier in the life history of lamprey, as biliary artesia occurs
during the transformation of filter-feeding larvae into parasites, and 2) females would also
develop glandular cells. A third hypothesis could be that males use bile acids to regulate the
increase in metabolism required for spawning. Recently, Watanabe et al. [37] suggested that bile
acids may increase energy availability in brown fat. Males possessing the ability to increase bile
acid metabolism may realize higher fitness resulting from an increase in energy. The glandular
cells may function to eliminate the additional bile acids, which can be toxic at high
concentrations [38]. Males may face stronger pressure to have higher energy than females as
they mature first, initiate nest construction, and need to put more investment into acquiring mates
where females invest heavily in gamete production [30]. The biological function of 3kPZS
synthesis and glandular cell development in silver lamprey merits further investigation.
Regardless of the biological function, the production and release of 3kPZS by
spermiating male silver lamprey may represent a physiological bias contributing to the
involvement of 3kPZS in the sexual signaling system of sea lamprey. For example, PZS is also
hypothesized to be a component of both the silver lamprey [39] and sea lamprey [40] migratory
cue. However, 3kPZS, not PZS, is the major component of the sea lamprey mating pheromone
[9]. Although natural-selection has shaped a preference for each compound, only 3kPZS
synthesis and release was found to be up-regulated by spermiating male silver lamprey. The
hypothesis that the increased production and release of 3kPZS created a physiological bias

62

towards signaling via 3kPZS rather than PZS, or another petromyzontid bile acid, seems
probable. The existing increase in 3kPZS production and release by spermiating males would be
amplified by increased fitness of males releasing higher quantities of 3kPZS, and the resulting
increase in females locating the spawning nest.
Although the data presented explain the origin of female preference for 3kPZS, the
mechanisms underlying male signaling with 3kPZS and the robust behavioral response in
females observed in sea lamprey remains unclear. Sexual conflict theory would predict that
males manipulate female preference to entice reproduction, a behavior often necessary due to the
sexual conflict arising from sexually asymmetric mating costs and divergent optimums [41]. The
exaggerated release of 3kPZS by male sea lamprey would have been driven by female’s existing
preference for 3kPZS and the resulting increase in mating opportunity for males. If male and
female mating optimums do not match and male signaling exploits female preference, female sea
lamprey would likely not have evolved to locate the source of 3kPZS. Female preference for
3kPZS may be mutually beneficial, suggesting that males and females possess similar mating
optimums. Originally the preference arose as an indicator of larval habitat, but as males realized
higher fitness with increased 3kPZS release, the females may also have realized higher fitness
with a fine-tuned attraction to 3kPZS.
In summary, I present data indicating that in the sea lamprey sexual communication
system, the female preference for male-released bile alcohol, 3kPZS, may have originated as a
result of a sensory bias. I infer that in the ancestral silver lamprey larval-released 3kPZS
functions as a migratory cue which guides females into habitable streams and male silver
lamprey have not evolved to manipulate the female preference by releasing 3kPZS at the high
rates observed in the sea lamprey [14]. Finally, I suggest that the chemical communication

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system of lampreys offers not only a useful model to study aquatic pheromones [16], but may
also prove useful in providing empirical evidence for communication theory [9].

64

REFERENCES

65

REFERENCES

1. Mandrioli, M., Malagoli, D. & Ottaviani, E. 2007 Evolution game: which came first, the
receptor or the ligand. Invertebrate Survival Journal 4, 51–54.
2. Endler, J. A. & Basolo, A. L. 1998 Sensory ecology, receiver biases and sexual selection.
Trends in Ecology & Evolution 13, 415–420. (doi:10.1016/S0169-5347(98)01471-2).
3. Andersson, M. & Simmons, L. W. 2006 Sexual selection and mate choice. Trends in Ecology
& Evolution 21, 296–302. (doi:10.1016/j.tree.2006.03.015).
4. Rodd, F. H., Hughes, K. A., Grether, G. F. & Baril, C. T. 2002 A possible non-sexual origin
of mate preference: are male guppies mimicking fruit? Proceedings of the Royal Society
B: Biological Sciences 269, 475–481. (doi:10.1098/rspb.2001.1891).
5. Basolo, A. L. 1990 Female preference predates the evolution of the sword in swordtail fish.
Science 250, 808–810. (doi:10.1126/science.250.4982.808).
6. Fuller, R. C., Houle, D. & Travis, J. 2005 Sensory bias as an explanation for the evolution of
mate preferences. The American Naturalist 166, 437–446. (doi:10.1086/444443).
7. Wyatt, T. D. 2003 Pheromones and animal behaviour. Cambridge: Cambridge University
Press. (doi: 10.2277/ 0521485266) .
8. Wyatt, T. D. 2009 Fifty years of pheromones. Nature 457, 262–263. (doi:10.1038/457262a).
9. Steiger, S., Schmitt, T. & Schaefer, H. M. 2011 The origin and dynamic evolution of
chemical information transfer. Proceedings of the Royal Society B: Biological Sciences
278, 970–979. (doi:10.1098/rspb.2010.2285).
10. Sorensen, P.W. and Stacey, N.E. 1999 Evolution and specializationof fish hormonal
pheromones. In: Advances in Chemical Signals in Vertebrates. pp. 15–47. Edited by R.E.
Johnston, D.Muller- Schwarze and P.W. Sorensen. Kluwer Academic/Plenum Publishers,
New York.
11. Murphy, C. A., Stacey, N. E. & Corkum, L. D. 2001 Putative steroidal pheromones in the
round goby, Neogobius melanostomus: olfactory and behavioral responses. Journal of
Chemical Ecology 27, 443–470. (doi:10.1023/A:1010376503197).
12. Kobayashi, M., Sorensen, P. W. & Stacey, N. E. 2002 Hormonal and pheromonal control of
spawning behavior in the goldfish. Fish Physiology and Biochemistry 26, 71–84.
(doi:10.1023/A:1023375931734).

66

13. Yambe, H., Kitamura, S., Kamio, M., Yamada, M., Matsunaga, S., Fusetani, N. &
Yamazaki, F. 2006 L-Kynurenine, an amino acid identified as a sex pheromone in the
urine of ovulated female masu salmon. Proceedings of the National Academy of Sciences
103, 15370–15374. (doi:10.1073/pnas.0604340103).
14. Li, W., Scott, A. P., Siefkes, M. J., Yan, H., Liu, Q., Yun, S. S. & Gage, D. A. 2002 Bile
acid secreted by male sea lamprey that acts as a sex pheromone. Science 296, 138–141.
(doi:10.1126/science.1067797).
15. Teeter, J. 1980 Pheromone communication in sea lampreys (Petromyzon marinus):
implications for population management. Canadian Journal of Fisheries and Aquatic
Sciences 37, 2123–2132. (doi:10.1139/f80-254).
16. Li, W. 2005 Potential multiple functions of a male sea lamprey pheromone. Chemical
Senses 30, i307–308. (doi:10.1093/chemse/bjh237).
17. Sorensen, P. W. & Hoye, T. R. 2007 A critical review of the discovery and application of a
migratory pheromone in an invasive fish, the sea lamprey Petromyzon marinus L.
Journal of Fish Biology 71, 100–114. (doi:10.1111/j.1095-8649.2007.01681.x).
18. Siefkes, M. J., Scott, A. P., Zielinski, B., Yun, S. S. & Li, W. 2003 Male sea lampreys,
Petromyzon marinus L., excrete a sex pheromone from gill epithelia. Biology of
reproduction 69, 125–132. (doi:10.1095/biolreprod.102.014472) .
19. Siefkes, M. J., Winterstein, S. R. & Li, W. 2005 Evidence that 3-keto petromyzonol
sulphate specifically attracts ovulating female sea lamprey, Petromyzon marinus. Animal
Behaviour 70, 1037–1045. (doi:10.1016/j.anbehav.2005.01.024).
20. Johnson, N. S., Yun, S. S., Thompson, H. T., Brant, C. O. & Li, W. 2009 A synthesized
pheromone induces upstream movement in female sea lamprey and summons them into
traps. Proceedings of the National Academy of Sciences 106, 1021–1026.
(doi:10.1073/pnas.0808530106).
21. Venkatachalam, K. 2005 Petromyzonol sulfate and its derivatives: the chemoattractants of
the sea lamprey. BioEssays 27, 222–228. (doi:10.1002/bies.20155).
22. Proctor, H. C. 1991 Courtship in the water mite Neumania papillator: males capitalize on
female adaptations for predation. Animal Behaviour 42, 589–598. (doi:10.1016/S00033472(05)80242-8).
23. Proctor, H. C. 1992 Sensory exploitation and the evolution of male mating behaviour: a
cladistic test using water mites (Acari: Parasitengona). Animal Behaviour 44, 745–752.
(doi:10.1016/S0003-3472(05)80300-8).
24. Potter I.C. 1980. The Petromyzoniformes with particular reference to paired species.
Canadian Journal of Fisheries and Aquatic Sciences 37:1595-1615.
67

25. Potter, I. C. & Gill, H. S. 2003 Adaptive radiation of lampreys. Journal of Great Lakes
Research 29, 95–112. (doi:10.1016/S0380-1330(03)70480-8).
26. Polkinghorne, C. N., Olson, J. M., Gallaher, D. G. & Sorensen, P. W. 2001 Larval sea
lamprey release two unique bile acids** to the water at a rate sufficient to produce
detectable riverine pheromone plumes. Fish Physiology and Biochemistry 24, 15–30.
(doi:10.1023/A:1011159313239) .
27. Neave, F. B., Mandrak, N. E., Docker, M. F. & Noakes, D. L. 2007 An attempt to
differentiate sympatric Ichthyomyzon ammocoetes using meristic, morphological,
pigmentation, and gonad analyses. Canadian Journal of Zoology 85, 549–560.
(doi:10.1139/Z07-032).
28. Li, K., Wang, H., Brant, C. O., Ahn, S. C. & Li, W. 2011 Multiplex quantification of
lamprey specific bile acid derivatives in environmental water using UHPLC-MS/MS.
Journal of Chromatography B 879, 3879–3886. (doi:10.1016/j.jchromb.2011.10.039).
29. Yeh, C-Y., Chung-Davidson, Y-W., Wang, H., Li, K., Li, W. Submitted. Adaptation to a
developmental biliary artesia in the sea lamprey.
30. Manion, P. J. & Hanson, L. H. 1980 Spawning behavior and fecundity of lampreys from the
upper three Great Lakes. Canadian Journal of Fisheries and Aquatic Sciences 37, 1635–
1640. (doi:10.1139/f80-211).
31. Siefkes, M. J. & Li, W. 2004 Electrophysiological evidence for detection and discrimination
of pheromonal bile acids by the olfactory epithelium of female sea lampreys (Petromyzon
marinus). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and
Behavioral Physiology 190, 193–199. (doi:10.1007/s00359-003-0484-1).
32. Cochran, P. A. & Lyons, J. 2004 Field and Laboratory Observations on the Ecology and
Behavior of the Silver Lamprey(Ichthyomyzon unicuspis) in Wisconsin. Journal of
Freshwater Ecology 19, 245–253. (doi:10.1080/02705060.2004.9664538).
33. Hardisty, M. W. and Potter, I. C. 1971. The behavior, ecology, and growth of larval
lampreys. In: The Biology of Lampreys. Volume 1. pp. 85–127. Edited by M.W. Hardisty
and I. C. Potter. Academic Press, London.
34. Morris, R. & Pickering, A. D. 1976 Changes in the ultrastructure of the gills of the river
lamprey, Lampetra fluviatilis (L.), during the anadromous spawning migration. Cell and
Tissue Research 173, 271–277. (doi:10.1007/BF00221380).
35. Pickering, A. D. & Morris, R. 1977 Sexual dimorphism in the gills of the spawning river
lamprey, Lampetra fluviatilis L. Cell and Tissue Research 180, 1–10.
(doi:10.1007/BF00227026).

68

36. Youson, J. H. 1993 Biliary atresia in lampreys. Advances in Veterinary Science and
Comparative Medicine. 37, 197–255.
37. Watanabe, M. et al. 2006 Bile acids induce energy expenditure by promoting intracellular
thyroid hormone activation. Nature 439, 484–489. (doi:10.1038/nature04330).
38. Hofmann, A. F. 1999 The continuing importance of bile acids in liver and intestinal disease.
Archives of Internal Medicine 159, 2647–2658. (doi:10.1001/archinte.159.22.2647).
39. Fine, J. M., Vrieze, L. A. & Sorensen, P. W. 2004 Evidence that petromyzontid lampreys
employ a common migratory pheromone that is partially comprised of bile acids. Journal
of Chemical Ecology 30, 2091–2110. (doi:10.1023/B:JOEC.0000048776.16091.b1).
40. Li, W., Sorensen, P. W. & Gallaher, D. D. 1995 The olfactory system of migratory adult sea
lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids
released by conspecific larvae. The Journal of General Physiology 105, 569–587.
(doi:10.1085/jgp.105.5.569).
41. Chapman T., Arnqvist G., Bangham J., Rowe L. 2003. Sexual conflict. Trends in Ecology &
Evolution 18, 41–47. (doi: 10.1016/S0169-5347(02)00004-6).

69