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THESIS

2004
379677457

 

 

 

LIBRARY
Michigan State This is to certify that the
University dissertation entitled

 

CHARACTERIZATION OF A MALE SEA LAMPREY SEX
PHEROMONE

presented by

MICHAEL J. SIEFKES

has been accepted towards fulﬁllment
of the requirements for the

PhD. degree in Fisheries and Wildlife

 

 

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V Major ProWr’s Signature

Date

 

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CHARACTERIZATION OF A MALE SEA LAMPREY SEX PHEROMONE
By

Michael J. Siefkes

A DISSERTATION
Submitted to
Michigan State University
In partial fulﬁllment of the requirements
For the degree of
DOCTOR OF PHILOSOPHY
Department of Fisheries and Wildlife

2003

ABSTRACT
CHARACTERIZATION OF A MALE SEA LAMPREY SEX PHEROMONE
By

Michael J. Sieﬂtes

Past studies have shown that spermiating male sea lampreys (Petromyzon
marinus) release a bile acid sex pheromone 7a, 12a, 24-tn'hydroxy-5a-cholan-3-one-24
sulfate (3-keto petromyzonol sulfate) that under laboratory conditions, induces search and
preference behaviors in ovulating females. However, the site of synthesis and excretion
of 3-keto petromyzonol sulfate, the electrophysiological potency and speciﬁcity of 3-keto
petromyzonol sulfate to the olfactory organs of females, and the effectiveness of 3-keto
petromyzonol sulfate and water conditioned with spermiating males in a natural
spawning environment have not been determined. In addition, whether bisazir-
sterilization of males (a current sea lamprey management technique) affects sex
pheromone function and thus competitiveness, has not been determined. In this
dissertation, results from behavioral assays, electro-olfactograms, biochemical analyses,
and immunucytochemistry showed that 3-keto petromyzonol sulfate is produced in the
liver and released exclusively through the gills of spermiating male sea lampreys. Also,
electro-olfactograms indicated that 3-keto petromyzonol sulfate is detected at a
concentration of 10"2 M and discriminated from other conspecific bile acids by the
olfactory organs of female sea lampreys. Furthermore, in-stream behavioral assays
demonstrated that 10'12 M 3-keto petromyzonol sulfate and water conditioned with

spermiating males (containing 2 x 10"2 M 3kPZS) function in a natural spawning stream

by attracting ovulating females 65 m to the source of each stimulus. Finally, behavioral
assays, electro-olfactograms, and mass spectrometry showed that bisazir-sterilized males
released a potent odorant (3-keto petromyzonol sulfate) that attracted ovulating females.
Combined, these results further elucidate the mechanisms whereby mature males attract
mature females and conﬁrm that spermiating males release a sex pheromone that
functions over relatively long distances. Overall, these results have the potential to
impact sea lamprey management by facilitating the pursuit of techniques that exploit sex
pheromone communication in sea lampreys and better our understanding of sex

pheromone communication in ﬁsh and vertebrates.

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Weiming Li and the members of my
graduate committee, Dr. Tom Coon, Dr. Doug Gage, Dr. Chris Goddard, and Dr. Dan
Hayes for their guidance and support. I would also like to acknowledge my co-authors of
the published papers contained in this dissertation; Mr. Roger Bergstedt, Dr. Weiming Li,
Dr. Alexander Scott, Mr. Michael Twohey, Dr. Sang-Seon Yun, and Dr. Barbara
Zielinski. Next, I am grateful to the staff of the US. Geological Survey, Hammond Bay
Biological station and the US. Fish and Wildlife Service, Marquette Biological Station
for supplying laboratory space, sea lampreys and technical assistance. Furthermore, I
would like to extend a special thanks to Dolly Trump and Lydia Lorenz for the use of
their private land adjacent to the Ocqueoc River and to the 19 undergraduate summer
assistants who have helped me through the years. Finally, I thank the Great Lakes

Fishery Commission for providing funding and enthusiastic support for this research.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ............................................................................................................ ix
INTRODUCTION ................................................................................................................ 1
Sex pheromones in ﬁsh ............................................................................................ 1
Sex Pheromones of the sea lamprey ......................................................................... 2
The sea lamprey as a model system ......................................................................... 3
Application of sex pheromones in sea lamprey management ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 5
Introduction of dissertation ...................................................................................... 6
Scientiﬁc signiﬁcance .............................................................................................. 9
References .............................................................................................................. 10
CHAPTER 1
MALE SEA LAMPREYS, Petromyzon marinas L., EXCRETE A SEX PHEROMONE
FROM GILL EPITHELIA ................................................................................................. 15
Abstract .................................................................................................................. 16
Introduction ............................................................................................................ 17
Materials and Methods ........................................................................................... 19
Collection and maintenance of animals ...................................................... 19
Collection of washings and extracts ........................................................... 19
Behavioral assays on water taken from the anterior and posterior regions
of spermiating males .................................................................................. 22
Electro-olfactographic recordings on water from the anterior and posterior
regions of Spermiating males ...................................................................... 23
Mass spectrometry analysis of water and urine from spermiating males_,23
ELISA of 3ketoPZS .................................................................................... 24
Immunocytochemistry for 3ketoPZS in the gills and liver of male sea
lamnreys ..................................................................................................... 25
Electron microsc0pic analysis of gill tissues .............................................. 27
Results .................................................................................................................... 28
Discussion .............................................................................................................. 39
Acknowledgements ................................................................................................ 43
References .............................................................................................................. 44
CHAPTER 2

ELECTROPHYSIOLOGICAL EVIDENCE FOR DETECTION AND
DISCRIMINATION OF PHEROMONAL BILE ACIDS BY THE OLFACTORY

EPITHELIUM OF FEMALE SEA LAMPREYS (Petromyzon marinas) ,,,,,,,,,,,,,,,,,,,,,,,,, 47
Abstract .................................................................................................................. 48
Introduction ............................................................................................................ 49
Materials and methods ........................................................................................... 53

EXperimental ﬁsh ....................................................................................... 53

 

Test Stimuli ................................................................................................ 53
Electro-olfactogram recording procedure ,,,,,,,,,,,,,,,,,,,,,,,,,,,, 54
Concentration-response relationships ........................................................ 55
Cross adaptation ......................................................................................... 56
Statistical analysis ...................................................................................... 57
Results .................................................................................................................... 59
Discussion .............................................................................................................. 65
Acknowledgements ................................................................................................ 71
References .............................................................................................................. 72
CHAPTER 3
A MALE SEA LAMPREY (Petromyzon marinus) SEX PHEROMONE THAT
ATTRACTS OVULATING FEMALES IN A SPAWNING STREAM ,,,,,,,,,,,,,,,,,,,,,,,,,,, 74
Abstract .................................................................................................................. 75
Introduction ............................................................................................................ 76
Materials and methods ........................................................................................... 78
Collection and maintenance of animals ..................................................... 78
Behavior in a Spawning stream .................................................................. 79
Independence tests of observed responses ................................................. 82
Results .................................................................................................................... 84
Discussion .............................................................................................................. 88
Acknowledgements ................................................................................................ 92
References .............................................................................................................. 93
CHAPTER 4
CHEMOSTERILIZATION OF MALE SEA LAMPREYS (Petromyzon marinus) DOES
NOT AFFECT SEX PHEROMONE RELEASE ............................................................... 95
Abstract .................................................................................................................. 96
Introduction ............................................................................................................ 97
Materials and methods ......................................................................................... 100
Collection and maintenance of animals ................................................... 100
Sterilization of male sea lampreys ........................................................... 100
Identiﬁcation of maturity ......................................................................... 101
Induction of Spenniation and ovulation ................................................... 101
Chemical stimuli used in experiments ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, _ 102

 

Experiment 1. In a two-choice maze, do ovulating females show
preference and searching behaviors when exposed to water conditioned

bisazir-sterilized, Spenniating males? ...................................................... 102
Experiment 2. In a spawning stream, are female sea lampreys attracted to
water conditioned with bisazir-sterilized, spermiating males? ,,,,,,,,,,,,,,,, 104

Experiment 3. Do female olfactory organs show the same
electrophysiological response to water conditioned with bisazir-sterilized
and nonsterilized, Spermiating males? ..................................................... 105

vi

Experiment 4. Do extracts of water conditioned with bisazir-sterilized and
nonsterilized, spermiating males contain the same pheromone molecule?
.................................................................................................................. 108

Results ............................................................
Discussion ......................................................
Acknowledgements ........................................

References

SUMMARY OF DISSERTATION ............................

APPENDIX
PERMISSION TO USE PUBLISHED MATERIAL

vii

109
120
124
125

128

131

LIST OF TABLES

Chapter 3

Table l. The distribution of ovulating and preovulating female sea lampreys swimming to
the treatment (TRT), control (CON), or staying at an intermediate position (INT) within a
section of a known sea lamprey spawning stream when water conditioned with
spermiating males (SMW) and 10'12 M 3-keto petromyzonol sulfate (synthetic 3kPZS)
treatments were used. The distributions of choices were signiﬁcantly different between
ovulating and preovulating females for each treatment suggesting an attraction by
ovulating females to the treatments (Fisher’s Exact Test, 2-Tail, P < 0.03).

Chapter 4

Table 1. Chemical stimuli from sterilized, spermiating male sea lampreys (Petromyzon
marinas) induced preference responses from ovulating females only. N: sample size.
Preference is the number of test subjects that spent proportionately more time after
stimulus introduction in treatment (scented) side. P-values were determined using a
Wilcoxon Signed Ranks Test (2-tailed) using indices of preference. NS: not signiﬁcant
(P > 0.10). Preference ratio is the mean ratio (standard deviation) of the time spent in
seconds in the treatment/control sides after stimulus introduction. *indicates ovulating
female tests using water conditioned with sterilized, spermiating males as the stimulus
source.

Table 2. Chemical stimuli from sterilized, spermiating male sea lampreys (Petromyzon
marinus) increased searching responses from ovulating females only. N: sample size.
Searching is the number of test subjects that spent proportionately more time swimming
after stimulus introduction in treatment (scented) side. P-values were determined using a
Wilcoxon Signed Ranks Test (2-tailed) using indices of preference. NS: not signiﬁcant
(P > 0.10). Searching ratio is the mean ratio (standard deviation) of the time in seconds
spent swimming in the treatment/control sides after stimulus introduction. *indicates
ovulating female tests using water conditioned with sterilized, spermiating males as the
stimulus source.

viii

LIST OF FIGURES

Chapter 1

Figure l. A) Schematic of the bisected aquarium used to collect washings from the
anterior and posterior regions of spermiating male sea lampreys. (a) indicates the anterior
and (b) the posterior chambers of the aquarium. A perforated acrylic tube (0), adjustable
mesh tube ((1), and latex gasket (e) were used to hold lampreys in place and prevent
contamination between the two chambers during washings. B) A female lamprey in the
bisected aquarium during a dye test (used to verify that there was no leakage from one
chamber to the other). The darker shading in the posterior chamber indicates the dye.

Figure 2. Electro-olfactogram responses of female sea lampreys to washings collected
from the anterior (ﬁlled diamonds; n = 6), posterior (ﬁlled triangles; n = 5), and whole
body (open circles; 11 = 6) of spermiating males. Responses are expressed as a percentage
of the response to a 10'5 M L-arginine standard. Vertical bars represent one standard
error.

Figure 3. Representative fast atom bombardment mass spectrometry analyses of extracts
from the anterior (A) and posterior (B) regions of spermiating males. The most abundant
ion in anterior extracts was at m/z 471, the same as the male sex pheromone and synthetic
3ketoPZS. Posterior extracts contained only trace amounts of this ion.

Figure 4. Mean release rate of 3ketoPZS estimated from washings collected from anterior
and posterior regions as well as directly from the gills of spermiating male sea lampreys.
Vertical bars, one standard deviation.

Figure 5. Confocal microscopy of pheromone immunocytochemistry against gill platelets
from presperrniating (A-C) and spermiating (D and E) male lampreys. A and D) Low-
power images; scale bar shown in A is 100 um. B,C, and E) High-power images; scale
bar shown in B is 15 um. All are images of scans in a single Z plane. B and C) the
interplatelet region sectioned following immunostaining from a single ﬁeld of view. B
was collected at 488 nm illumination, and the immunostaining is on the platelet surface
along the basal region of the platelet. C was collected through transmitted light detection
of bright-ﬁeld microscopy.

Figure 6. Subcellular localization of pheromone immunostaining. A-C) The interplatelet
region of a spermiating male gill ﬁlament sectioned following immunostaining; scale bar
shown in A is 15 pm. A and B) A single ﬁeld of view. A is viewed at 488 nm and B
through bright-ﬁeld microscopy. C) Immunoreactivity in the supranuclear region of
epithelial cells in the interplatelet epithelium. D and E) Pheromone immunoreactivity in
the platelet and interplatlet regions following lipid solubilization with 0.1% Triton-X100;

scale bar shown in D is 100 pm.

Figure 7. Transmission electron microscopy of gill platelets from a spermiating male. A-
C are the same magniﬁcation; scale bar in A is 0.5 pm. A) A low microvillar platelet
cell. These cells contain small electron lucent vesicles (thin arrows), large electron
Opaque granules (short thick arrows), and smooth endoplasmic reticulum (sER). B) A
cuboidal cell from the basal region of the interplatelet region has a dome-shaped apical
surface, rough endoplasmic reticulum (rER), Golgi apparatus (G), electron dense vesicles
(arrow), dense core vesicles (dcv), and electron lucent vesicles (elv). C) The
supranuclear cytoplasm of an interplatelet cell contains electron opaque granules (arrows)
and smooth endoplasmic reticulum (sER). D) The perinuclear cytoplasm of an
interplatelet cell with granules of varying sizes and electron density (arrows) and tubular
cistemae of smooth endoplasmic reticulum (sER); scale bar in D is 1 pm.

Figure 8. Pheromone immunostaining in the liver of spermiating (A) and presperrniating
(B) male sea lampreys; scale bar in A is 50 um. Both images were acquired using a X60
oil immersion objective with the same laser power, gain, iris diameter constant, and black
level to avoid artifacts from differences in exposure and settings.

Chapter 2

Figure 1. Molecular structure of sea lamprey bile acids. A 3a, 7a, 12a, 24-tetrahydroxy—
5a-cholan-24-sulfate; Petromyzonol sulfate; PZS, B 7a, 12a, 24-trihydroxy-50t-cholan-
3-one-24-sulfate; 3 keto-petromyzonol sulfate; 3kPZS, C 3a, 7a, 120t-trihydroxy-50t-
cholan-24-oic-acid; Allocholic acid; ACA, and D 7a, l2a-dihydroxy-5a-cholan-3-one-
24-oic-acid; 3 keto-allocholic acid; 3kACA. P28 and ACA are released by larval sea
lampreys and 3kPZS and 3kACA are released by spermiating male sea lampreys. Notice
that P28 and 3kPZS possess a sulfate group, while ACA and 3kACA possess a carboxyl
group on carbon 24. Also notice that P28 and ACA possess a hydroxyl group whereas
3kPZS and 3kACA possess a carbonyl group on carbon 3.

Figure 2. A Semi-logarithmic plots of electro-olfactogram (EOG) concentration-
responses to sea lamprey bile acids (allocholic acid, ACA; petromyzonol sulfate, P28; 3
keto-allocholic acid, 3kACA; 3 keto-petromyzonol sulfate, 3kPZS). B Semi-logarithmic
plots of EOG concentration-responses to water conditioned with spermiating males
(SMW) and 3kPZS. C and D expanded view of responses in A and B, respectively
showing response threshold concentrations. The response threshold for a given odor is
the lowest concentration that elicits a response signiﬁcantly greater than the control
(students t-test, P<0.05). The asterisks (*) denote the response threshold of 3kPZS in C
and 3kPZS and SMW in D. The carrot (A) denotes the response threshold of the other
bile acids in C. Mean response magnitudes are presented as a percentage of the response
elicited by a 10‘5 M L-arginine standard solution. Vertical bars represent one standard
error. Numbers by the abbreviations indicate sample sizes

Figure 3. Results of cross-adaptation experiments, grouped by the ﬁve adapting stimuli:
A) 3 keto-petromyzonol sulfate, 3kPZS; B) 3 keto-allocholic acid, 3kACA; C)
petromyzonol sulfate, PZS; D) allocholic acid, ACA; E) water conditioned with
spermiating males, SMW. Mean percent initial responses (PIR) are shown with

horizontal lines representing one standard error. For each adapting stimulus, the response
to self-adapted controls is underlined. Number symbols (#) signify that a particular test
stimulus was completely adapted to control levels, meaning the test stimulus PIR was not
different from the self-adapted control PIR (P>0.05, Dunnett’s test). Asterisks (*) signify
that a particular test stimulus was partially adapted, meaning the test stimulus PIR was
different from the self-adapted control PIR (P<0.05, Dunnett’s test), but the test stimulus
response during adaptation was less than the response before adaptation (responses in mV
not shown; P<0.05, paired t-test). Carrots (A) signify that a particular test stimulus was
not adapted, meaning the test stimulus response during adaptation was the same as the
response before adaptation (responses in mV not shown; P>0.05, paired t-test). Numbers
by the bars indicate sample sizes. The numbers by the abbreviations are the logarithmic
values of the molar concentrations for the tested stimuli. NT, not tested.

Chapter 3

Figure 1. The spawning stream layout used in radio tracking experiments. C1 and C2
indicate points in which water conditioned with spermiating males or 3-keto
petromyzonol sulfate and control odorants were randomly introduced. A indicates the
acclimation cage in which females were held before testing. Arrows indicate water ﬂow.
The dashed line represents the downstream block net used to prevent lampreys from
exiting the study site.

Chapter 4

Figure 1. (a) Representative electro-olfactogram (EOG) responses of female sea
lampreys (Petromyzon marinas) to chemical stimuli from sterilized, spermiating males
(SSM, diamonds), non-sterilized, spermiating males (SM, open circles), and non-
sterilized, pre-spermiating males (PSM, ﬁlled circles). Std designates the response to a
10‘5 M L-arginine standard and Can the response to a blank water control. Numbers
along the x-axis indicate the logarithmic value of the dilution from the original
conditioned water collected by holding one male in 10 l for 4 h. (b) Female sea lamprey
EOG dose-response relationships to SSM (N = 8), SM (N = 8), and PSM (N = 4)
chemical stimuli. Responses are measured as a percentage of the response to a 10'5 M L-
arginine standard. Vertical bars represent one standard error.

Figure 2. (a) Representative electro-olfactogram (EOG) responses of female sea
lampreys (Petromyzon marinas) to chemical stimuli from sterilized spermiating male
(SSM) and nop-gterilized spermiating male (SM) before (white bars) and during (shaded
bars) adaptatiofr to a SSM chemical stimuli. Std designates the response to a 10'5 M L-
arginine standard and Con the response to a blank water control. Female EOG responses
to a 10'5 M L-arginine standard, SSM and SM chemical stimuli both before and during
adaptation to SSM (b) and SSM (c) chemical stimuli were measured. Vertical bars
represent one standard deviation.

Figure 3. Negative and positive fast atom bombardment mass spectrometry (10 KV)
spectrum of (a) extracts of water conditioned with non-sterilized spermiating male sea

xi

lampreys (Petromyzon marinas) and (b) extracts of water conditioned with sterilized
spermiating males.

xii

INTRODUCTION

Sex pheromones in ﬁsh

A pheromone is composed of “substances that are excreted to the outside by an
individual of the same species in which they release a speciﬁc reaction, for example a
deﬁnite behavior or developmental process” (Karlson and Luscher 1959). Many ﬁsh
species release potent odorants that function as sex pheromones during ﬁnal maturation
(Reviews: Scott and Vermeirssen 1994; Stacey et al. 1994). Speciﬁcally, sex
pheromones in ﬁsh have been shown to stimulate vitellogenesis (Van Weerd and Kamen
1998) and ovulation (Dmitrieva and Ostroumov 1986), induce courtship behavior
(Ostroumov and Dmitrieva 1990) and synchronize the release of sperm and eggs (Stacey
and Hoursten 1982). To date the majority of sex pheromone research in ﬁsh has focused
on female sex pheromones. A good example is the goldﬁsh, Carassius auratus sex
pheromone system where at least three compounds have been identiﬁed and function to
synchronize goldﬁsh spawning physiology and behaviors (Sorensen and Scott 1994).
Other female pheromones in ﬁsh have also been characterized in species such as rainbow
trout, Oncorhynchus mykiss (Newcombe & Hartman 1973, Scott et al. 1994); Atlantic
salmon, Salmo salar (Waring et al 1996); zebraﬁsh, Brachydanio reri (Lambert et al.
1986; Van Den Hurk and Resink 1992); and loach, Misgurnus anguillicaudatus
(Kitamura and Ogata 1990).

Male sex pheromones, although less extensively studied than female sex

pheromones in ﬁsh, have been reported in numerous species such as Ictalurid catﬁsh

(Todd et al. 1967; Richards 1974); steelhead, Oncorhynchus mykiss (Newcombe &
Hartman 1973); blenny, Blennius pavo (Laumen et al. 1974); ﬁve belontiid species (Lee
& Ingersoll 1979); black goby, Gobiusjozo (Colombo et al. 1980, 1982); and Paciﬁc
herring, Clupea harengus pallasi (Stacey & Hourston 1982; Sherwood et al. 1991;
Carolsfeld et al. 1997). These pheromones have been found to attract mature females to
mature males or to induce physiological changes such as ovulation in female

conspeciﬁcs.

Sex pheromones of the sea lamprey

Mature male sea lampreys have long been suspected of releasing sex pheromones.
In France, where sea lampreys were collected for food, ﬁshermen used mature males as
bait to lure females to their traps (F ontaine I93 8). When placed in a two-choice maze,
mature females spent more time in the compartment where washings from sexually
mature males had been introduced (Teeter 1980). Also, in a two-choice maze anchored
in a spawning stream, mature females were attracted to mature males (Li 1994). This
makes sense in the context of sea lamprey reproductive biology, because it is usually the
male who initiates nest building, a process joined later by one or more females and males
(Applegate 1950). In addition, it has been demonstrated that spermiating males release a
potent odorant that could be a sex pheromone (Li 1994; Bjerselius et al. 1996; Sieﬂtes
2000).

Recent research indicates that spermiating male sea lampreys release a sex

pheromone 7a, 12a, 24-trihydroxy-5a-cholan-3-one-24-sulfate (3 keto-petromyzonol

sulfate, or 3kPZS), which elicits search and preference responses from ovulating females
in a two-choice maze and natural spawning stream (Sieﬂtes 2000; Li et al. 2002).
Sperrniating males were also found to release another bile acid, 7a, 120t-dihydroxy-50t-
cholan-3-one-24-oic-acid (3 keto-allocholic acid, 3kACA; Yun et al. 2003a); however, it
has not been experimentally tested for any sex pheromone function. Pre-spermiating

males were not attractive to ovulating females and did not release 3kPZS or 3kACA.

The sea lamprey as a model system

The sea lamprey offers a unique animal model for studies of sex pheromone
communication in vertebrates. This species represents Cephalaspidomorphi, one of the
two extant groups of Agnathan, or jawless ﬁshes, and thus is among the most primitive
vertebrate species available for examination of pheromone-induced behaviors.
Understanding the functions of its sex pheromone will offer new insights into the
evolution of pheromone communication among vertebrate animals. Another unique
feature of the sea lamprey is that it is monorhynic or has only a single olfactory organ,
which precludes the possibility of simultaneous comparison of odor intensities across
space. This makes it a great challenge to localize and approach odor sources. Yet it has
been demonstrated that sea lampreys rely on odorants to identify conspeciﬁc individuals,
prey, spawning streams, and mates. Presumably sea lampreys have evolved innate
locomotion patterns which, when displayed upon detection of relevant odorants, bring sea

lampreys to the source of odorants. By examining the components of sex pheromones

and the behavioral patterns elicited by them, an understanding of behavioral mechanisms
whereby animals localize or approach odor sources can be developed.

The sea lamprey also offers a useful model for studying speciﬁcity of chemical
communication throughout life history. Sea lampreys develop through three distinct life
stages, all regulated to some degree by odorants. Growth of larval sea lampreys appears
to be regulated by metabolites of conspeciﬁcs (Mallatt 1983). After a radical
transformation, juvenile sea lampreys enter a parasitic stage during which they rely on
odorants to localize host ﬁsh (Kleerekoper and Mogensen 1963; Kleerekoper and van
Erkel 1960). In early spring, sea lampreys start to enter certain rivers to spawn. Recent
studies Show that adults rely on a larval odorant to select suitable spawning streams
(Teeter 1980; Moore and Schleen 1980; Li 1994; Bjerselius et al. 2000; Polkinghome et
al. 2001; Vrieze and Sorensen 2002). Finally, once sea lampreys are on the spawning
grounds, spermiating males release a pheromone that attracts ovulating females to their
nest (Sietkes 2000; Li et al. 2002).

Finally, the sea lamprey offers an intriguing model for the use of sex pheromones
in management of vertebrate pest species. Sex pheromones have been used extensively
to control undesirable insects (Carde and Minks 1995), but similar techniques have not
been applied to vertebrate pests because either the pheromones or their functions have not
been identiﬁed or where they have, pheromones have not been shown to function in
natural conditions or over distances great enough to be effective. The spermiating male
sea lamprey sex pheromone is the ﬁrst male sex pheromone identiﬁed in ﬁsh and one of

the few sex pheromones identiﬁed in vertebrates. Moreover it is the ﬁrst bile acid sex

pheromone identiﬁed in ﬁsh and has been shown to function under natural conditions

over distances as great as 65 meters (Li et al. 2002).

Application of sex pheromones in sea lamprey management

The Sea Lamprey is a non-indigenous species and an often-lethal parasite of the
larger ﬁshes in the upper Great Lakes (eg. Bergstedt and Schneider 1988; Kitchell 1990).
The sea lamprey invasion caused economic and ecological tragedy in terms of their
impact on the ﬁsh communities in the Great Lakes (Smith and Tibbles 1980). Sea
lamprey management is essential to restore and maintain the Great Lakes ecosystem and
a sustainable ﬁshery (Bergstedt and Schneider 1988). Currently lampricides, barriers,
trapping, and sterile male releases are used to control sea lamprey populations (Klar and
Young 2002). However, sea lampreys continue to be a signiﬁcant source of mortality for
large and medium sized ﬁsh in the Great Lakes (Bergstedt and Schneider 1988; Kitchell
1990). For this reason, and the fact that current control techniques have the potential to
be costly and environmentally damaging (Smith and Tibbles 1980; Lamsa et al. 1980),
the development of additional means of sea lamprey management needs to occur.

At the ﬁrst Sea Lamprey International Symposium held in 1979 (Smith 1980), it
was proposed that pheromones, if identiﬁed, could be used in the management of sea
lamprey populations in the Great Lakes (Teeter 1980). Potential management strategies
using pheromones are based on the premise that natural odorants produced by sea
lampreys can inﬂuence or disrupt spawning behaviors and ultimately, reproduction.

Spawning sea lampreys congregate in known streams across the Great Lakes basin and

are targeted efﬁciently. Sea lampreys have well-developed olfactory organs
(Kleerekoper 1972) and large olfactory bulbs relative to brain size (Stoddart 1990). This
anatomically dominant system is highly sensitive to a unique variety of compounds (Li
1994; Li et al. 1995) that regulate prey searching, migration and mating (Kleerekoper and
Mogensen 1959; 1963; Teeter 1980; Bjerselius et al. 2000). Management strategies
based on exploitation of the sea lamprey sense of smell and odor-induced behaviors are
likely to be effective, efﬁcient and environmentally sound.

Several strategies have been proposed to exploit the sex pheromone
communication of sea lampreys for management purposes (Li et al. 2003; Twohey et al.
2003). One way is to develop techniques to enhance the biosynthesis and release of
3kPZS in sterilized males in hopes that they successfully obtain more mates. A second
way is to use a synthetic copy of 3kPZS for attraction and annihilation of females. Other
possible ways include exploiting biochemical and physiological processes to disrupt the
signaling system (either preventing males from releasing sex pheromone or preventing
females from detecting it) and using a possible physiological priming sex pheromone to

disrupt ﬁnal maturation in both males and females.
Introduction of dissertation

Although many questions have been answered regarding the sex pheromone
communication system of sea lampreys, much more needs to be known about the
production, release, detection and function of the spermiating male sex pheromone before

it can be incorporated into sea lamprey management. The goal of this dissertation was to

expand upon the knowledge already known about a male sea lamprey sex pheromone (Li
et al. 2002). The chapters of this dissertation will cover the four objectives outlined
below, which were designed to ﬁll in critical gaps in the knowledge of this pheromone
communication system.

Objective 1: Determine the release site of sex pheromone in spermiating male sea
lampreys

It has long been thought that the urine was the source of sex pheromones in male
sea lampreys (Teeter 1980). Alternatively, recent research has suggested that 3kPZS is
released from the gills of spermiating males (Li et al. 2002; You et al. 2003a), however,
there has been no concrete evidence for the gill release pathway suggested in the recent
studies. If the production and release sites of 3kPZS are known, it may provide a means
to disrupt sex pheromone production and release, which could reduce a male’s
reproductive success. Also, information on the production site of 3kPZS may assist in
ﬁnding its synthetic pathway and ultimately may yield a cheaper way to manufacture this
compound in mass quantities, which currently is quite costly.

Objective 2: Determine if bile acids released by conspecific larvae and adult sea
lampreys possess different odor qualities and can be distinguished from one another by
female olfactory organs.

In a natural stream, there are many bile acid compounds that are very similar in
structure to 3kPZS. Larval sea lamprey (also present in spawning streams) release 3a,
7a, 12a, 24-tetrahydroxy-5a-cholan-24-sulfate (petromyzonol sulfate, P28) and 3a, 7a,
120t-trihydroxy-5a-cholan-24-oic-acid (allocholic acid, ACA), bile acids associated with
adult sea lamprey migration. Also, spermiating male sea lampreys release another bile
acid, 3kACA that may also act as a sex pheromone or is a component of the male sex

pheromone. The ability to distinguish between these odorants of similar chemical

structure seems challenging for sea lampreys and has not been experimentally
determined. This could provide information on the complexity of the sea lamprey
olfactory system and may provide insight into developing sea lamprey management
strategies. Also, information on the potency of 3kPZS will provide clues on the
concentrations at which this sex pheromone could be used in sea lamprey management.

Objective 3: Determine if the male sex pheromone is eﬂective in a natural spawning
environment.

Traps baited with sex pheromones have been a common practice to control
undesirable insects (Carde and Minks 1995), but similar techniques have not been applied
to vertebrate pests because either the signals and their functions have not been identiﬁed
or where they have, pheromones have not been shown to attract target individuals in
natural conditions or over distances great enough to be effective. Water conditioned with
spermiating males and synthetic 3kPZS have not yet been demonstrated to function in a
spawning environment. Information on the effectiveness of male sex pheromone in a
natural spawning stream will provide valuable preliminary data on the feasibility of
developing pheromone-baited traps for integrated sea lamprey management.

Objective 4: Determine if bisazir-sterilization of male sea lampreys inhibits sex
pheromone release and function

An important premise behind the sterile male release technique is that the
sterilization process does not negatively affect the mating competency of sterilized
individuals (Knipling 1964). It has not yet been determined if bisazir-sterilization
inhibits sex pheromone release and function, but if it does, the ability of sterilized males

to attract mates would be reduced. Determining the effect of sterilization on the male sex

pheromone will provide justiﬁcation for the use of the sterile male release technique and

precludes the creation of pheromone-enhanced sterilized males.

Scientiﬁc significance

Results from this study may lead to the development of new concepts and
techniques for integrated sea lamprey management and provide the ﬁrst use of
pheromones in the management of a vertebrate species. In addition to the direct
relevance to sea lamprey management, results advance our understanding of pheromone
communication in ﬁsh and vertebrates. This is the ﬁrst male sex pheromone and ﬁrst bile
acid sex pheromone identiﬁed in ﬁsh and has been shown to function at low
concentrations in a natural environment. Moreover this study offers the ﬁrst example that
gills contain the machinery to and do actively release a sex pheromone. In the long run,
information on how this pheromone is produced and released and how adult sea lampreys
smell bile acids produced by conspeciﬁcs will bring beneﬁts outside of sea lamprey

management that are not immediately apparent.

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13

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14

CHAPTER 1

MALE SEA LAMPREYS, Petromyzon marinas L., EXCRETE A SEX
PHEROMONE FROM GILL EPITHELIA

Sieﬂtes, M.J., Scott, A.P., Zielinski, B., Yun, S.-S., and Li, W. 2003. Male sea lampreys,
Petromyzon marinas L., excrete a sex pheromone from gill epithelia. Biology of
Reproduction, 69:125-132.

 

15

ABSTRACT

During the period when they are producing sperm, male sea lampreys
(Petromyzon marinus L.) release a sex pheromone 7a, 12a, 24-trihydroxy-50t-cholan-3-
one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and
preference behaviors in ovulating females. In this study, we conducted a series of
experiments to demonstrate that release of this pheromone into water takes place
exclusively through the gills. In a behavioral maze, water conditioned with the anterior
region of spermiating males induced an increase of search and preference behaviors in
ovulating females. Similar behavior was not elicited by water conditioned by the
posterior region. The anterior region washings and whole body washings from
spermiating males also elicited large and virtually identical electro-olfactogram responses
from female sea lampreys, while the posterior washings produced negligible responses.
Further, mass spectrometry and immunoassay conﬁrmed that virtually all the 3ketoPZS
released into water was through the gills. Immunocytochemistry revealed some gill
epithelial cells and hepatocytes from spermiating males contained dense immunoreactive
3ketoPZS, but not those from prespermiating males. These results demonstrate that
3ketoPZS is released through the gill epithelia, and suggest that this pheromone or its

precursor may be produced in the liver.

16

INTRODUCTION

Spermiating male sea lampreys (a “spermiating” lamprey is one from which milt
[spermatozoa plus seminal ﬂuid] can be expressed by gentle manual pressure),
Petromyzon marinus L. release a bile acid, 7a, 12a, 24-trihydroxy-5a-cholan-3-one-24-
sulfate (3 keto-petromyzonol sulfate, 3ketoPZS), which acts as an attractant for ovulating
females [1]. Although this sex pheromone has been identiﬁed and its function
demonstrated, its mode of synthesis and excretion remains elusive. Bile acids are
typically produced in the liver, secreted into the gall bladder, and excreted through the
intestine along with feces [2, 3]. Larval sea lampreys appear to have evolved this route as
well [4, 5]. However, this same route is not available to adult sea lampreys, which lack
gall bladders and bile ducts [6].

In river lamprey Lampetraﬂuviatilis, the gills of spawning males contain large
glandular cells [7, 8] that have been postulated to “secrete some substance of sexual
signiﬁcance” [7]. More recently, we showed that water from the anterior region of
spermiating males (bathing the gills) contained far more immunoreactive 3ketoPZS than
water from the posterior region [9] and suggested that gills may mediate the release of
this pheromone compound [1]. However, there was no proof that this compound
emanated from the gills rather than from some other part of the anterior region and
certainly no direct link to the glandular cells. Further, there was no corroborating
chemical and biological evidence from any other procedure that 3ketoPZS was indeed

emanated mainly from the anterior region.

17

The main hypothesis that we address in the present study is that the male
pheromone is released into the water via the glandular cells of the gills. Our expectations
are 1) that the results of ELISA [9], which showed that 3ketoPZS was released largely
from the anterior region of spermiating males, can be conﬁrmed by mass spectrometry,
electro-olfactogram and behavior induction in ovulating females; 2) that water emitted by
gills (before it touches any other tissues) contain 3ketoPZS at a level comparable to that
estimated from anterior body washings; 3) that the unusual glandular cells in the gills will
stain immunocytochemically with antibodies to 3ketoPZS; and 4) that the gills of

spermiating males have many more of these cells than the gills of prespermiating males.

18

MATERIALS AND METHODS

Collection and maintenance of animals

This research was approved by the Michigan State University, All University
Committee on Animal Use and Care and complied with all federal and state laws,
policies, and rules for the humane use of laboratory animals in research. Adult sea
lampreys were collected from tributaries to lakes Huron and Michigan by the staff of the
US. Fish and Wildlife Service, Marquette Biological Station, Marquette, MI. The
animals were transported to the main laboratory at the US. Geological Survey,
Hammond Bay Biological Station, Millersburg, MI. Males and females were separated
and held in ﬂow-through tanks (1000 L) with Lake Huron water at temperatures ranging
from 7°C to 20°C. Males and females were checked periodically for spermiation and
ovulation according to the criteria and procedures reported [10]. Any spermiating and

ovulating individuals were separated into two other tanks, respectively.

Collection of washings and extracts

Washings from the anterior and posterior regions of spermiating males were
collected using a bisected, acrylic aquarium (Figure 1). A divider with a hole to
accommodate a sea lamprey’s head was ﬁxed in the middle of the aquarium to make two
separate chambers. The hole was lined with a latex gasket that, when the male was in

place, prevented water from ﬂowing between the two chambers. A perforated acrylic

l9

 

 

I{'-""'I'¥ """"""""" 7|

b---—i—h —————————————— &

 

 

 

 

 

 

 

 

 

Figure l. A) Schematic of the bisected aquarium used to collect washings from the
anterior and posterior regions of spermiating male sea lampreys. (a) indicates the anterior
and (b) the posterior chambers of the aquarium. A perforated acrylic tube (c), adjustable
mesh tube (d), and latex gasket (e) were used to hold lampreys in place and prevent
contamination between the two chambers during washings. B) A female lamprey in the
bisected aquarium during a dye test (used to verify that there was no leakage from one
chamber to the other). The darker shading in the posterior chamber indicates the dye.

20

tube was mounted on one side of the hole to immobilize the anterior region. The
posterior region was held in ﬂexible plastic mesh that was tightened according to the size
of the male.

During a washing, a spermiating male was anaesthetized with tricaine methane
sulfonate (MS-222; Argent Chemical Laboratories, Redmond, WA) and placed headﬁrst
through the gasket/divider, making sure all the gills were located in the forward chamber.
The plastic mesh tube around the posterior region was then tightened. The anterior
chamber was ﬁlled with 7 L of water, the latex gasket inspected for leaks, and then the
posterior chamber ﬁlled with 7 L of water. Each chamber was aerated, and the male was
held this way for 1 h. At the end, all the water was siphoned into buckets and either used
directly in experiments or extracted as previously reported [1]. For extractions, water
was pre-ﬁltered with No. 3 Watman ﬁlter paper (Millipore Corp., Bedford, MA) and then
drawn through activated Sep-Pak octadecylsilane cartridges (Waters, Milford, MA).
These were then washed with distilled water, eluted with methanol, and stored at -80°C.

To prove that there was no leakage across the latex gasket in the bisected
aquarium, two experiments were performed both before and alter the washing collection
period. In the ﬁrst, males were secured in the aquarium. A dye was then added to one
chamber and the other checked visually after 1 h (Figure 1B). The aquarium was then
drained and the experiment repeated, adding dye to the other chamber, checking for
leakage in the other direction. In the second experiment, females, known not to release
3ketoPZS [1, 9], were placed in the aquarium and 1 mg of synthetic 3ketoPZS was
introduced into one of the chambers. After 1 h, water samples were taken from both

chambers and analyzed for 3ketoPZS by ELISA [9]. Again, the aquarium was drained

21

and the experiment repeated, adding synthetic 3ketoPZS to the other side, checking for

leakage in the other direction.

Behavioral assays on water taken from the anterior and posterior regions of
spermiating males

The sex pheromone released by spermiating males induces preference and search
behaviors from ovulating females [1, 10]. Therefore, we measured these two types of
characteristic behavioral responses from ovulating females to chemical stimuli from the
anterior and posterior regions of spermiating males using the identical apparatus and
protocols developed in the previous studies [1, 10]. Brieﬂy, preference behavior was the
amount of time spent in either side of a two-choice maze, while search behavior was the
amount of time spent swimming at the head of either side of a two-choice maze. In each
test, the behavior of a single female was video recorded before and after the introduction
of washings from either the anterior or posterior regions of spermiating males into the
odor chamber of the side of the maze chosen randomly by the toss of a coin. Because the
same females were used to test both washings, the order of washing presentation was also
randomized. The washings were delivered at 75 ml/min using a peristaltic pump. Tests
were conducted between 0700 and 1700 in water temperatures that ranged from 12 °C to
24 °C. Preference and search behaviors were scored by naive observers and the data

analyzed with a two-tailed Wilcoxon signed rank test [1, 10, 11].

22

Electro-olfactographic recordings on water from the anterior and posterior regions
of spermiating males

Synthetic and natural 3ketoPZS induce strong electro-olfactographic (EOG)
responses from adult female sea lampreys [1], as does water conditioned by spermiating
male sea lampreys [10, 12]. Using EOG recording on females, we determined the
olfactory potency of washings from the anterior and posterior regions and the whole body
of spermiating males and established concentration-response relationships, according to
established procedures [10, 13]. For each recording, a 10'5 M L-arginine standard was
pulsed into the olfactory epithelium of a female and the EOG response measured to
establish a baseline of electrical activity. Next, blank control water was introduced and
the response measured to conﬁrm the absence of odorants from the clean water source
used to perfuse the olfactory epithelium (and also to mix the test odorants). Increasing
concentrations of test odorants (starting at 106 dilution) were then introduced and the
responses measured. Measuring the response to the L-arginine standard and blank
controls concluded each trial. The epithelium of each female was allowed to recover for
at least 3 min between stimuli and each concentration of an odorant was tested at least
twice on a female. The magnitude of EOG responses was measured [14] and expressed

as a percentage of the L-arginine standard [13].

Mass spectrometry analysis of water and urine from spermiating males

Methanol extracts of washings (l L) taken from the anterior and posterior regions

of spermiating males were dried down under a stream of oxygen-free nitrogen gas at

23

45°C, reconstituted in chloroform and subjected to fast atom bombardment mass
spectrometry (FAB MS, 10KV) in both negative and positive ionization modes.

To determine whether 3ketoPZS was present in the urine, spermiating males were
anesthetized with an MS-222 solution and a catheter was inserted into the urogenital pore
[15]. Urine was collected into a 50 ml container that was emptied periodically and stored
at -80°C until analysis. The urine was passed through a Sep-Pak and the eluate dried

down and subjected to FAB MS as described previously.

ELISA of 3ketoPZS

The procedure for ELISA of 3ketoPZS has been described [9]. The antibody
shows 100% cross reaction with 7d, 12a, 24-trihydroxy-50t-cholan-3-one (3 keto-
petromyzonol, 3kPZ) and 7a, l2a-trihydroxy-5a-cholan-3—one-24-oic acid (3 keto-
allocholic acid, 3kACA). However, the former was not found in washing extracts from
spermiating males [9] and the latter was present at a ratio of only 1:25
(3kACA:3ketoPZS; [16]). The anterior and posterior washings from spermiating males
were diluted 1:25 with assay buffer before ELISA.

ELISA was also carried out on water collected directly from the gills of
spermiating males. To do this, a male was anesthetized with metomidate hydrochloride
(Syndel, Vancouver, BC, Canada), immobilized with gallamine triethiodide (Sigma
Chemical Co., St. Louis, MO), and placed in a ﬂow-through trough. Water ﬂowed
through the mouth and exited the gills at an average rate of 431 ml/min. Water was

pipetted directly off the gills and placed into a beaker every three minutes for 0.5 h,

24

making a total of 10 individual samples for each spermiating male. Parts of each sample
were stored at -80°C, and 100 ml of each sample were extracted using a Sep-Pak, and the
eluate was also stored at -80°C until ELISA analysis.

Hourly release rates of 3ketoPZS were calculated for the anterior region washings
and water collected directly from the gills. For the anterior washings, the 3ketoPZS level
of water was multiplied by the total volume used in the anterior portion of the aquarium
(7 L). For water collected directly from the gills, the average 3ketoPZS level of water for
each spermiating male was multiplied by the ﬂow of water across the gills (431 ml/min)

and then by 60 min.

Immunocytochemistry for 3ketoPZS in the gills and liver of male sea lampreys

Five spermiating and seven prespermiating males were killed with an overdose of
MS-222. The gills and liver were removed, immersed in either Zamboni’s ﬁxative (2%
paraformaldehyde and 1.2% saturated picric acid in 0.1 M phosphate buffer [PB]) or 4%
paraformaldehyde (in PB) and stored at 4°C. As the male pheromone is a steroidal lipid,
whole gill ﬁlaments were mounted to minimize the loss of cellular lipids [17]. Each gill
pouch was placed under a dissection microscope, and single gill ﬁlaments were removed.
Throughout the immunostaining procedure, the gill ﬁlaments were agitated mildly at 4°C.
The single gill ﬁlaments were rinsed 3 times over 1 h with either PB or with PB plus
0.1% Triton X-100 (PB-TX) for 30 min. The omission of Triton-X from the
immunostaining protocol served to assist in keeping the cellular membranes and lipid-

containing subcellular structures intact. Nonspeciﬁc binding was blocked with 5%

25

normal goat serum in PB for 20 min, drained off and exchanged for primary antibody
(from four rabbits: codes 184, 185, 286, 285 [9]) diluted with 0.1 M PB or 0.1 M PB-TX
(1:1000 to 1:5000) and incubated for 24 h. The tissue was then rinsed three times over 1
h with 0.1M PB, followed by a 3-h incubation in Alexa 488 goat anti-rabbit IgC, which
excites at 488 nm (1:100; Molecular Probes, Eugene, OR); rinsed; and mounted in
glycerol or viewed directly with a 40X water immersion lens. Following
immunostaining, some ﬁlaments were sectioned with a vibratome (Leica Microsystems,
Wetzlarrn, Germany) to allow for viewing of the interplatelet region. The immunostained
ﬁlament was embedded in 5% agarose (American Bioanalytical, Natick, MA) and
sectioned at a thickness of 150 um. All preparations were viewed using a BioRad 1024
Confocal Microscope (Bio-Rad, Hercules, CA) with the following settings: laser power,
10%; iris, 1.5 mm; gain, 1000X; black level, 0. Fixed liver samples were embedded in
5% agarose, sectioned using a vibratome at a thickness of 200 um, and stained for
3ketoPZS by the procedure used for the gill whole mounts (see previous discussion).
Liver samples were viewed with a 60X oil immersion lens using the same settings.

A preadsorption control experiment was conducted using a 10-fold molar excess,
relative to IgC concentration, of antigen (3ketoPZS) to antibody dilution. The
3ketoPZS/antibody mixture was incubated for 24 h at 4°C, with mild agitation, then
centrifuged at 100,000 g for 30 min (Sorvall RC M120 GX, Kendro, Asheville, NC). The
supernatant was used in place of the primary antibody in the previously described
immunocytochemical protocol. The preadsorption control did not contain any
immunostaining. Negative controls were also conducted using preimmune sera for

antisera from rabbits 184 and 185 [16]. Tissues processed with these preimmune sera

26

were unstained. In addition, a control with the primary antibody omitted was included in

each experiment.

Electron microscopic analysis of gill tissues

The gill tissues from spermiating and prespermiating male sea lampreys were
ﬁxed in buffered 2.5% glutaraldehyde, and then in 1% 0504. After dehydration, ﬁxed
tissues were embedded in Spurtol resin. Ultrathin sections (90 nm) were cut, and then
stained with uranyl acetate and lead citrate. The sections were examined in Philips CM10

electron microscope.

27

RESULTS

Dye tests (n = 2) and ELISA measurements of water spiked with 3ketoPZS (n =
2) revealed that there was no detectable exchange of water between the two chambers of
the bisected aquarium (data not shown). The total time spent and the time spent
searching in each side of the two-choice maze before odorant introduction showed that
ovulating females were not biased to either side (Student t-test, P > 0.10; data not
shown). When water from the anterior region of spermiating males was introduced into
the maze, ll of 14 ovulating females spent more of their total time (Wilcoxon signed
rank test, P < 0.01) and six of seven spent more time searching (Wilcoxon signed rank
test, P < 0.01) in the side to which the water was introduced. There were no signiﬁcant
increases or decreases in total time spent (seven of 14; Wilcoxon signed rank test, P >
0.10; n = 14) or time spent searching (two of seven; Wilcoxon signed rank test, P > 0.10;
n = 7) in the experimental side of the maze when the posterior region water was
introduced.

In EOG experiments, the mean response to 10'5 M L-arginine was 0.5 mV (SE =
0.09 mV; n = 6). The anterior region washings (n = 6) of spermiating males were far
more potent than those of the posterior region (11 = 5) at equivalent dilutions (Figure 2),
but were equipotent to those from whole spermiating males (11 = 6). The detection
threshold for the posterior region washings was approximately 1:100 (v/v), whereas that
for the anterior region and whole-body chemical stimuli was approximately 1:10,000

(v/v).

28

500 '

400 ‘

300 ‘

200 e

100 "

 

 

Response (% L-arginine standard)

 

Log dilution

Figure 2. Electro-olfactogram responses of female sea lampreys to washings collected
from the anterior (ﬁlled diamonds; n = 6), posterior (ﬁlled triangles; n = 5), and whole
body (open circles; n = 6) of spermiating males. Responses are expressed as a percentage
of the response to a 10'5 M L-arginine standard. Vertical bars represent one standard
error.

29

Negative FAB MS analyses showed that the most abundant ion in extracts of
water from the anterior region of spermiating males was at m/z 471 (n = 6; Figure 3A),
the same as synthetic 3ketoPZS [1]. At positive mode, the base peak was at m/z 473.
FAB MS detected only trace amounts of this molecule in the extracts of water from the
posterior regions (11 = 6, Figure BB) and in extracts of spermiating male urine (n = 4, data
not shown).

The mean concentration of immunoreactive 3ketoPZS in water collected from the
anterior regions of spermiating males was 54 ng/ml (n = 13) whereas that from the
posterior regions was 0.8 11ng (n = 13). On average, the total release rate of the anterior
region was 378 ug/h (Figure 4). Also, immunoreactive 3ketoPZS was found in water
collected directly off the gills of ﬁve spermiating males; however, these concentrations
showed considerable variation (Figure 4). On average, the release rate of 3ketoPZS
estimated from water collected directly off the gills was 308 pg/h. There was no
signiﬁcant difference between release rates estimated from the anterior region water and
gill water (Stundent t-test, P > 0.10).

The gill ﬁlaments were comprised of gill platelets (lamellae, Figure 5A) covered
by low cuboidal platelet cells, with cuboidal to columnar shaped cells in interplatelet
regions. In prespermiating males, the pheromone immunoreactivity was weak and
diffuse on the platelet surface and granular in interplatelet epithelial cells (Antibody 286;
Figure 5A-C). In gill tissue from spermiating males, subpopulations of platelet cells were
strongly immunoreactive to the pheromone antibodies (antibody 185; Figure 5D and E).
This staining was absent from the nucleus (Figure 5E, 6A). Both platelet and inter-

platelet epithelial cells contained supranuclear regions that were intensely

30

50-
40-
30'
20‘

10-

Relative abundance (%)

 

400

Relative abundance (%)

 

471

 

Figure 3. Representative fast atom bombardment mass spectrometry analyses of extracts
from the anterior (A) and posterior (B) regions of spermiating males. The most abundant
ion in anterior extracts was at m/z 471, the same as the male sex pheromone and synthetic
3ketoPZS. Posterior extracts contained only trace amounts of this ion.

31

 

 

 

 

 

 

 

 

 

0 I r ———_‘
Anterior Gills Posterior

 

Figure 4. Mean release rate of 3ketoPZS estimated from washings collected from anterior
and posterior regions as well as directly from the gills of spermiating male sea lampreys.
Vertical bars, one standard deviation.

32

 

Figure 5. Confocal microscopy of pheromone immunocytochemistry against gill platelets
from prespermiating (A-C) and spermiating (D and E) male lampreys. A and D) Low-
power images; scale bar shown in A is 100 um. B,C, and E) High-power images; scale
bar shown in B is 15 pm. All are images of scans in a single Z plane. B and C) the
interplatelet region sectioned following immunostaining from a single ﬁeld of view. B
was collected at 488 nm illumination, and the immunostaining is on the platelet surface
along the basal region of the platelet. C was collected through transmitted light detection
of bright-ﬁeld microscopy.

33

 

Figure 6. Subcellular localization of pheromone immunostaining. A-C) The interplatelet
region of a spermiating male gill ﬁlament sectioned following immunostaining; scale bar
shown in A is 15 pm. A and B) A single ﬁeld of view. A is viewed at 488 nm and B
through bright-ﬁeld microscopy. C) Immunoreactivity in the supranuclear region of
epithelial cells in the interplatelet epithelium. D and E) Pheromone immunoreactivity in
the platelet and interplatlet regions following lipid solubilization with 0.1% Triton-X100;
scale bar shown in D is 100 um.

34

immunoreactive to the pheromone antibodies (antibody 286; Figure 6A-C). These
immunopositive cells were cuboidal along the platelet base regions (Figure 6A) and
columnar in the interplatelet regions (Figure 6C). In all samples, pheromone
immunoreactivity was stronger and clearer when Triton-X was omitted from
immunostaining procedure than in samples treated with Triton-X (antibody 185; Figure
6D and E). The fact that the immunoreactivity decreased and blurred following
treatment with the lipid solubilizing detergent is consistent with the lipid nature of the
pheromone.

Transmission electron microscopy of the gill epithelial cells revealed low
microvillar cuboidal platelet cells (Figure 7A) with small electron lucent vesicles, larger
electron opaque granules (0.3 pm), round mitochondria, rough and smooth endoplasmic
reticulum, and Golgi apparatus. In the basal platelet region and the interplatelet region,
the cuboidal cells (Figure 7B) had a dome-shaped apical surface, smooth endoplasmic
reticulum, rough endoplasmic reticulum, electron dense vesicles, dense core vesicles, and
electron lucent vesicles. The supranuclear region of low columnar interplatelet cells
(Figure 7C) contained electron opaque granules and smooth endoplasmic reticulum. The
perinuclear cytoplasm of interplatelet cells (Figure 7D) contained granules of varying
sizes and electron density and numerous vesicular and tubular cistemae of smooth
endoplasmic reticulum. These ultrastructural characteristics Show that these cells are
active in lipid metabolism and store secretory products.

In spermiating male liver, the immunostaining (antibody 285) was strong and
diffuse in hepatocyte cytoplasm, and strong in widespread cytoplasmic granules (Figure

8A). The liver of pre-spermiating males displayed pheromone immunoreacitivity

35

 

Figure 7. Transmission electron microscopy of gill platelets from a spermiating male. A-
C are the same magniﬁcation; scale bar in A is 0.5 pm. A) A low microvillar platelet
cell. These cells contain small electron lucent vesicles (thin arrows), large electron
opaque granules (short thick arrows), and smooth endoplasmic reticulum (sER). B) A
cuboidal cell from the basal region of the interplatelet region has a dome-shaped apical
surface, rough endoplasmic reticulum (rER), Golgi apparatus (G), electron dense vesicles
(arrow), dense core vesicles (dcv), and electron lucent vesicles (elv). C) The
supranuclear cytoplasm of an interplatelet cell contains electron opaque granules (arrows)
and smooth endoplasmic reticulum (sER). D) The perinuclear cytoplasm of an
interplatelet cell with granules of varying sizes and electron density (arrows) and tubular
cistemae of smooth endoplasmic reticulum (sER); scale bar in D is 1 pm.

36

 

Figure 8. Pheromone immunostaining in the liver of spermiating (A) and prespermiating
(B) mal sea lampreys; scale bar in A is 50 um. Both images were acquired using a X60
oil immersion objective with the same laser power, gain, iris diameter constant, and black
level to avoid artifacts from differences in exposure and settings.

37

(antibody 285) that was weak and difﬁrse, with scattered intensely stained cytoplasmic
granules (Figure 8B). Therefore, the pheromone is present in higher levels in the

spermiating male liver than in the liver of the prespermiating male.

38

DISCUSSION

The data from the present study clearly support our hypothesis that the male sex
pheromone 3ketoPZS is released through gills, most likely via the glandular cells that
appear in the gills of spermiating males. The ﬁrst subhypothesis, that 3ketoPZS is
released almost exclusively from the anterior portion of spermiating males, is
unequivocally supported by data from our behavioral assays, EOG recording, ELISA and
mass spectrometry analyses. In the two-choice maze, washings collected from the
anterior regions of spermiating males induced in ovulating females the preference and
search behaviors that are characteristic of those induced by 3ketoPZS [1] whereas the
washings from the posterior region did not. This is corroborated by electrophysiological
results that the anterior region water is equipotent to the whole body water in stimulating
the female olfactory organ and is about 100 times more potent than the posterior region
water. Chemically, both ELISA and mass spectrometry indicate that 3ketoPZS is present
in large amounts in the anterior region water samples but in negligible amounts in the
posterior region water and urine samples. These results conﬁrm and expand on the
previous ELISA results that the anterior region washings contain large amounts of
3ketoPZS [9].

The EOG and ELISA data also indicate that the 3ketoPZS released from the gills
accounts for all the 3ketoPZS released by the whole animal, supporting the second
subhypothesis. The anterior region washings induce EOG responses at virtually the same
magnitude as the whole washings did over a range of dilutions that spans four orders of

magnitude. The average release rate from the anterior region, 378 jig/h, is close to the

39

previously reported whole body release rate of c. 500 pg/h [9]. Most importantly, the
washings collected directly from the gills contain large amounts of 3ketoPZS (~308 ug-h'
'), which can account for the majority of the 3ketoPZS release estimated from the
washings collected from the anterior region. These results support the gill-release
hypothesis set forth in our previous study [1].

Finally, our third subhypothesis is clearly supported by immunocytochemical
experiments that show the presence of 3ketoPZS in cells of both the liver and gills. It
appears likely that the pheromone, a sulfate- and ketone- containing bile acid derivative,
moves into the water from the platelet and interplatelet cells of spermiating males.
Diffuse immunolabeling on the surface of platelet cells of both stages may be due to the
secreted pheromone adhering to the cell surface of these cells. The localization of
pheromone immunoreactive granules in the interplatelet cells of prespermiating males
suggests that pheromone-containing cells in the platelet epithelium ﬁrst appear in the
interplatelet region during the prespermiating phase and then only in the platelet region
during spermiation. It is likely that 3ketoPZS is at least a part of the “substance of sexual
signiﬁcance” [7].

Our results clearly implicate the involvement, but do not illustrate the explicit role
of hepatocytes in biosynthesis of 3ketoPZS. The granules with strong pheromone
immunoreactivity demonstrate that hepatocytes contain either 3ketoPZS, 3kACA, or
3kPZ, or all of them because the primary antibody used for immunocytochemical staining
cross-reacts with all three of these compounds. This conforms with the discovery that

PZS, a larval bile acid, is produced in the liver [5] and that liver is the exclusive organ for

bile acid synthesis [18]. It is possible that 3ketoPZS is synthesized in the liver, released

40

into circulation, and taken up by the platelet and interplatelet cells. Alternatively, the gill
cells may take up a precursor synthesized in the liver and modify it into 3ketoPZS.
Smooth endoplasmic reticulum, a subcellular site for steroid synthesis is widespread in
the platelet and interplatelet cells. Granules of varying electron density that are
prominent in transmission electron micrographs may be the site of the granular
localization of pheromone immunoreacitivity of the interplatelet cells. Large lipid
structures with electron lucent centers, previously observed in Type I male glandular cells
of river lamprey ﬁxed in osmium tetroxide [8], were absent from sea lamprey. This
difference may be due to the differing ﬁxation protocols, with stabilizing aldehyde
preﬁxation being included in the present study but absent from the earlier study.

In bony ﬁshes, all pheromones identiﬁed to date are steroids or prostaglandins - or
sulfated or glucuronidated forms of these compounds [19-22]. In the case of free
steroids, there is evidence that they, like the lamprey pheromone, are mainly excreted into
the water via the gills [23, 24]. However, the mechanism whereby they do so appears to
be passive diffusion [23, 25]. There is no evidence for specialized cells such as those that
we have demonstrated in lampreys. The release rates of free steroids by bony ﬁshes are
also considerably lower than those of 3ketoPZS by spermiating lampreys [1, 26]. In
contrast to free steroids, sulfated steroids in teleosts and elasmobranches do not pass
through the gills [23, 25]. Their main site of release appears to be the urinary bladder
[15, 24]. Although the urinary bladder has been thought as a source of sex pheromones
in the sea lamprey [27], none has yet been ﬁrmly identiﬁed. The main reason for

making these comparisons between vertebrate classes is to underline our belief the

41

mechanism whereby male sea lampreys release 3ketoPZS is an active one, with the
specialized cells that appear in the gill epithelium of spermiating males as “pumps.”

To rely on gills to broadcast 3ketoPZS possibly represents the evolution of a
system mainly to extend the active space of the pheromone signal. Such a system would
certainly be advantageous for spermiating male lampreys, which construct nests in sites
where water ﬂows at 0.5 to 1.5 m/sec [28, 29] and probably have to rely heavily on this
pheromone to attract ovulating females from downstream [1, 27].

Bile acids, and their sulfate esters such as 3ketoPZS, are produced in large
quantities by hepatocytes of vertebrates [18]. The transportation of 3ketoPZS from liver
to gills is potentially very efﬁcient because, in the lamprey, hepatic veins carry blood
directly to the heart and all blood from the heart passes immediately through the gills
[30]. It has been estimated that in a 100-g river lamprey, the relative area of the gills is
600 m2/ g [31]. If the relative surface area in the sea lamprey were similar, its gills
would provide an enormous surface area with specialized glandular cells, through which
the respiratory activity generates continuous ﬂow to facilitate exchange of the pheromone
molecule with the environment.

In conclusion, our experiments demonstrate that the spermiating male sea lamprey
sex pheromone, 3ketoPZS, is released through the gills. Although liver and glandular
cells are clearly implicated in the production and release of 3ketoPZS, their explicit roles
need to be examined at the molecular level to elucidate the mechanistic processes

whereby this pheromone molecule is produced and released.

42

ACKNOWLEDGEMENTS

We thank the staffs of U. S. Fish and Wildlife Service, Marquette Biological
Station, Marquette, MI and the U. S. Geological Survey, Hammond Bay Biological
Station, Millersburg, M1 for collecting sea lampreys and providing space to conduct
experiments for this study; Dolly Trump and Lydia Lorenz for the use of their private
land to conduct behavioral experiments; Andrea Belanger for her assistance with
immunocytochemistry; Sally Burns for her assistance with electron microscopy; and

Beverly Chamberlin for her assistance with mass spectrometry.

43

10.

11.

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Yun SS, Sieﬂces MJ, Scott AP, Li W. Development and application of an ELISA
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Sieﬂces MJ, Bergstedt RA, Twohey MB, Li W. Chemosterilization of male sea
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46

CHAPTER 2

ELECTROPHYSIOLOGICAL EVIDENCE FOR DETECTION AND
DISCRIMINATION OF PHEROMONAL BILE ACIDS BY THE OLFACTORY
EPITHELIUM OF FEMALE SEA LAMPREYS (Petromyzon marinas)

Sieﬂtes, M.J. and Li, W. In press. Electrophysiological evidence for detection and

discrimination of pheromonal bile acids by the olfactory epithelium of female sea
lampreys (Petromyzon marinus). Joum_al of Compaﬂtive Physiology A.

47

ABSTRACT

Electro-olfactograms were used to determine sensitivity and speciﬁcity of
olfactory organs of female sea lampreys (Petromyzon marinas) to four bile acids: 3-keto
petromyzonol sulfate and 3-keto allocholic acid from spermiating males and
petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are
thought to function as a mating pheromone and larval bile acids as a migratory
pheromone. The response threshold was 10‘12 M for 3-keto petromyzonol sulfate and 10’
'0 M for the other bile acids. At concentrations above 10'9 M, the sulfated bile acids
showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile
acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol
sulfate and water conditioned with spermiating males induced similar concentration-
response curves and response thresholds. Cross adaptation experiments demonstrated
that the sulfated and non-sulfated bile acids represent different odors to the olfactory
epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate
represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and
allocholic acid represent the same odor. Results indicate that male-speciﬁc bile acids are
potent and speciﬁc stimulants to the female olfactory organ, supporting the previous

hypothesis that these bile acids function as a pheromone.

48

INTRODUCTION

Bile acids are potent stimulants for both the gustatory and olfactory systems of
many ﬁsh species (Hara 1994, Li et al. 1995; Michel and Lubomudrov 1995). Initially, it
was proposed that the bile acids are appealing candidates for salmon pheromones due to
their structural diversity and stability (Doving et al. 1980; Stabell 1987). Furthermore,
electrophysiological studies demonstrated that an odotopic map of responses to bile acids
and amino acids are represented in different regions of olfactory bulbs of chars
(Salvelinus alpinus) and graylings ( T hymallus thymallys) (Doving et al. 1980).
Behavioral studies in Atlantic salmon (Salmo salar) also showed that intestinal extracts,
which are known to contain bile acids, induced strain-speciﬁc preference and searching
behaviors (Stabell 1987). Indeed, several experiments indicated that a number of bile
acids induced behavioral responses in ﬁsh (Jones and Hara 1985; Hellstrom and Doving
1986; Sola and Tosi 1993; Hara 1994). More recently, it has been shown that bile acid
proﬁles of lake trout (Salvelinus namaycush) are largely inﬂuenced by sex and
maturation stage (Zhang et al. 2001) and that bile of female rainbow trout (Oncorhynchus
mykiss) contains a pheromone (Vermeirssen and Scott 2001). However, no speciﬁc bile
acids have been identiﬁed as pheromones in teleost ﬁsh.

Recently, the sea lamprey (Petromyzon marinas) has emerged as an efﬁcient
animal model for studies of bile acids as possible pheromones because it is the only
species in which speciﬁc bile acids have been linked to speciﬁc pheromone functions.
Biochemical and behavioral studies have shown that sea lamprey at different life stages

release speciﬁc bile acids that induce characteristic behaviors in adults (Bjerselius et al.

49

2000; Li et al. 2002). This species experiences three stages during its life cycle. The sea
lamprey inhabits tributary streams during the larval stage, enters the Atlantic Ocean or
large lakes (e. g. the Laurentian Great Lakes) to feed after metamorphosis into the
parasitic stage and returns to streams in the adult stage to spawn and die (Hardisty and
Potter 1971). Larval sea lampreys release two bile acids, petromyzonol sulfate (PZS) and
allocholic acid (ACA) (Haslewood and Tokes 1969; Li et a1. 1995; Polkinghome et al.
2001; Figure 1A and B), that are potent stimulants to olfactory organs (Li et al. 1995; Li
and Sorensen 1997) and induce locomotion behaviors in adults of the same species in a
laboratory maze (Bjerselius et a1. 2000; Vrieze and Sorensen 2001). Furthermore, adult
males, after onset of spermiation, release two bile acids, 3-keto petromyzonol sulfate
(3kPZS; Li et a1. 2002; Figure 1C) and 3-keto allocholic acid (3kACA; Yun et al. 2003;
Figure 1D). 3kPZS has been shown to induce increased swimming activity in ovulating
females and their ultimate attraction (Li et al. 2002). The function of 3kACA has not
been determined and is suspected to be a minor component of the male pheromone (Yun
et al. 2003).

These four bile acids present a physiological challenge for the olfactory system of
adult sea lampreys that use them as possible pheromones. Sea lamprey adults spawn in
rapids where current velocity typically reaches 1 m s'1 (Applegate 1950). Bile acids
released by males would be rapidly diluted. Moreover, the adult male bile acids, 3kPZS
and 3kACA, only differ from their larval counterparts (PZS and ACA) by having a
carbonyl, as apposed to a hydroxy at carbon 3 (Figure 1). Yet larvae inhabit spawning
streams year round (Moore and Schleen 1980) and release PZS and ACA when

spermiating males release 3kPZS and 3kACA to attract ovulated females. It is essential

50

 

Figure 1. Molecular structure of sea lamprey bile acids. A) 3a, 7a, 12a, 24-tetrahydroxy-
5a-cholan-24-sulfate; Petromyzonol sulfate; PZS, B) 7a, 12a, 24-tn'hydroxy-50t-cholan-
3-one-24-sulfate; 3 keto-petromyzonol sulfate; 3kPZS, C) 3a, 7a, 12a-trihydroxy-50t-
cholan-24-oic-acid; Allocholic acid; ACA, and D) 7a, 12a-dihydroxy-5a-cholan-3-one-
24-oic-acid; 3 keto-allocholic acid; 3kACA. PZS and ACA are released by larval sea
lamprey and 3kPZS and 3kACA are released by spermiating male sea lampreys. Notice
that PZS and 3kPZS possess a sulfate group, while ACA and 3kACA possess a carboxyl
group on carbon 24. Also notice that PZS and ACA possess a hydroxyl group whereas
3kPZS and 3kACA possess a carbonyl group on carbon 3.

51

for mature female sea lampreys to be able to distinguish male bile acids from larval ones.
Further, it has yet to be shown directly that 3kPZS and 3kACA stimulate the olfactory
epithelium of adult females.

In this study, our objectives were to determine electrophysiologically 1) the
potency of 3kPZS and 3kACA to the olfactory organs of adult females, and 2) if the
olfactory epithelium of females discriminates among the four bile acids. Our
electrophysiological data from this study indicate that 3kPZS, demonstrated to function
as a male pheromone (Li et al. 2002), represents a highly potent and distinct odor to adult

female sea lampreys.

52

MATERIALS AND METHODS

Experimental Fish

Adult sea lampreys were collected from tributaries to lakes Huron and Michigan
between April and July 2000-2002 by the staff of the US. Fish and Wildlife Service,
Marquette Biological Station, Marquette, Michigan, USA. Lampreys were transported to
the main laboratory at the US. Geological Survey, Hammond Bay Biological Station,
Millersburg, Michigan USA. Females were separated from males and held in ﬂow-
through tanks ( 1000 L) with chilled Lake Huron water at temperatures ranging from 6°C
to 8°C. Chilled water slows senescence, preserving measurable olfactory responsiveness

throughout the spawning season.

Test Stimuli

The olfactory epithelia of female sea lampreys were exposed to solutions of L-
arginine (Sigma Chemicals, St. Louis, Missouri, USA), four bile acids (ACA, PZS,
3kACA, 3kPZS; Toronto Research Chemicals, Canada; all >97% pure), and water
conditioned with spermiating males (SMW). Stock solutions of bile acids were made at
concentrations of 10'3 Molar (M) using de-ionized water or methanol and stored at —

20°C. A 10'2 M L-arginine standard stock solution was made with de-ionized water

every week and stored at 4°C.

53

To collect SMW, males were held in ﬂow-through tanks (200 L; 15 males per
tank) with heated Lake Huron water at temperatures ranging from 15°C to 18°C. These
temperatures were used to promote spermiation. Males were checked for spermiation
according to the criteria set forth by Siefkes et al. (2003a). Water was conditioned with
spermiating male sea lampreys by holding individual males for 4 h in polyethylene
buckets ﬁlled with 10 L of Lake Huron water. These buckets were aerated and kept in a
water bath of 18°C. The SMW were either used immediately in or stored at -80°C for
electro-olfactographic analyses. The SMW were measured for 3kPZS concentration
using an enzyme-linked immunosorbent assay (ELISA; Yun et al. 2002), but were not

measured for the other bile acids used in this study or for any other compounds.

Electro-olfactogram recording procedure

The same source of Lake Huron water was used to maintain the animal, collect
male lamprey odor, dilute bile acids, and perfuse the naris during recording.
Female sea lampreys were tested for olfactory sensitivity to the four bile acids and SMW
described above. Electro-olfactogram (EOG) recording was performed as described by
Li et al. (1995). Brieﬂy, sea lampreys were anesthetized with an intramuscular injection
of metomidate hydrochloride (3 mg kg'1 body weight; Syndel, Vancouver, British
Columbia, Canada), immobilized with an intramuscular injection of gallamine
triethiodide (150 mg kg'1 body weight; Sigma, St Louis, Missouri, USA) and placed in a
water-ﬁlled trough. The head of the female remained above the water and the gills were

supplied with aerated water. The olfactory lamellae were then exposed and perfused with

54

water. Differential electrical potential between the skin surface and the sensory epithelia
in response to each test stimulus were recorded using two Ag/AgCl electrodes (type EH-
18, World Precision Instruments, Sarasota, Florida, USA) ﬁlled with 3 M potassium
chloride and bridged with 8% gelatin:0.9% saline — ﬁlled glass capillary tubes. The
recording electrode was placed between two lamellae and was adjusted to maximize the
response to the L-arginine standard while minimizing the response to a blank water
control when the reference electrode was placed on the skin near the naris. Electrical
signals were ampliﬁed and digitized by a Power Lab (ADI Instruments, Castle Hill, NSW

2154, Australia) and displayed on a computer.

Concentration-response relationships

Stock solutions of odors were diluted in Lake Huron water immediately before
testing. To determine concentration-response relationships, a 10'5 M L-arginine standard
was introduced into the olfactory epithelium of a female for 5 seconds and the E06
response measured to establish a reference of electrical activity (Li et al. 1995). Next
blank water control was introduced and the response measured to conﬁrm the absence of
response from the water supply. Increasing concentrations of bile acids starting at 10'13
M and SMW were then introduced and the responses measured. Measuring the response
to the L-arginine standard and blank water control again at the end of the dilution series
concluded each trial. The epithelium of each female was allowed to recover for at least 3

min between stimuli, and each concentration of the test stimuli was assayed at least twice.

55

EOG response magnitudes from females were measured in mV and expressed as a

percentage of the response to the L-arginine standard.

Cross adaptation

Cross adaptation, developed by Caprio and Byrd (1984), was used to compare the
EOG response to a test stimulus before and during adaptation to an adapting compound
using a protocol by Li and Sorensen (1997). In a given trial, baseline olfactory EOG
responses of females to blank water control (Control A), an L-arginine standard and test
stimuli (SMW and all four bile acids; ‘Initial Response’) were recorded. The test stimuli
were used at concentrations that elicited approximately equipotent olfactory responses at
about 100% of the L-arginine standard. During adaptation, the olfactory epithelium of a
female was continually exposed to the adapting stimulus for 5 min after which a 5 second
application of the same adapting stimulus was tested, ﬁrst at the concentration used in the
adaptation (‘Control B’) and then at twice the concentration used for the adaptation
(‘Self-adapted Control’). Then the other test stimuli were tested in the adapting solution
(‘Adapted Response’). Each test was interspersed with 5 second applications of the L-
arginine standard and Control B to conﬁrm the responsiveness of the female. Switching
the adapting stimulus back to blank water completed the trial. The epithelium of the
female was allowed to recover for 30 min and then the female was tested again using
another adapting stimulus. Cross adaptation data were expressed as Percent Initial
Response (PIR) using the following formula adapted from Caprio and Byrd (1984) and Li

and Sorensen (1997),

56

 

R _ (Adapted Response — Control B) X
(Initial Response — Control A)

100

where a larger PIR indicates less cross-reactivity between olfactory receptor mechanisms
or separate receptor sites and a low PIR indicates more cross reactivity or shared receptor

sites and/or a common signal transduction pathway.

Statistical analysis

Statistical analysis system (SAS) was used to conduct all analyses. In
concentration-response experiments, responses were visually compared. The lowest
concentration at which a stimulus elicited a response larger than the blank water control
(Student’s t-test) was considered to be its response threshold.

In cross adaptation experiments, a paired t-test was used to determine if responses
to a test stimulus during adaptation were signiﬁcantly different than the responses to the
same test stimulus before adaptation. All PIR data were subsequently analyzed to
directly detect cross-reactivity by subjecting all PIR data to a two-way analysis of
variance (ANOVA). If the main effect was found to be signiﬁcant, the PIR data was
divided into ﬁve groups according to adapting stimulus. For each group, responses of
adapted epithelia to test stimuli were analyzed by a one-way ANOVA to determine the
affect of the adapting stimulus. Again, if the main effect was signiﬁcant, the signiﬁcance
of the adapting effect for each test stimulus was determined by comparing all PIRS in the
group with the PIR of the self-adapted control using Dunnett’s test, which tests for

differences between several treatments and a single control.

57

The following categories were used to classify cross adaptation responses; 1) not
adapted, meaning responses during adaptation were not signiﬁcantly different from the
initial response (paired t-test, P>0.05); 2) partially adapted, meaning responses during
adaptation were signiﬁcantly less than the initial responses (paired t-test, P<0.05), but the
PIR were signiﬁcantly greater than the control (Dunnett’s, P<0.05); and 3) adapted to
control levels, meaning PIR were not signiﬁcantly different than the control (Dunnett’s,

P>0.05).

58

RESULTS

All chemicals tested were stimulatory to the olfactory epithelium of female sea
lampreys. The mean response to the L-arginine standard was 0.859 :E 0.04 mV (mean i
SE). Concentration-response relationships of the four bile acids were plotted together as
percentages of the L-arginine standard (Figure 2A). All responses to bile acids ranged
from —9 % to 640 %, and the blank water control elicited a mean response of 7.9 i 6.5 %
(mean i SE). 3kPZS and PZS had similar concentration-response curves that were
exponential in shape with steep slopes between 10"0 and 10"5 M. The response threshold,
or the lowest concentration that elicited a response Signiﬁcantly greater than the blank
water control, for 3kPZS was 10'12 M (31.4 i 3.2 %, mean :1: SE; Student’s t-test, P<0.01;
Figure 2C) and for PZS was 10'10 M (26.8 i 9.6%, mean i SE; Student’s t-test, P<0.01;
Figure 2C). 3kACA and ACA had similar concentration-response curves that were linear
in shape from the response threshold of 10''0 M (37.8 :L- 20.5%, mean :1: SE; Student’s t-
test, P<0.01 and 30.0 i 7.5%, mean i SE; Student’s t-test, P<0.01, respectively; Figure
2C) to 10’6M, the highest concentration tested. The concentration-response curves for
3kACA and ACA were shallower than the curves generated with 3kPZS and PZS.

When comparing the concentration-response curves of SMW and 3kPZS, the
curve for SMW was plotted according to the concentration of 3kPZS measured in the
SMW samples using ELISA (Yun et al. 2002). The concentration-response relationship
of SMW had an exponential shape similar to the 3kPZS curve (Figure 23). Also, both
SMW and 3kPZS had response thresholds of 10'12 M (16.2 i 3.2 %, mean i SE;

Student’s t-test, P=0.05 and 31.4 i 3.2 %, mean i SE; Student’s t-test, P<0.01,

59

Figure 2. A) Semi-logarithmic plots of electro-olfactogram (EOG) concentration-
responses to sea lamprey bile acids (allocholic acid, ACA; petromyzonol sulfate, PZS; 3
keto-allocholic acid, 3kACA; 3 keto-petromyzonol sulfate, 3kPZS). B) Semi-
logarithmic plots of EOG concentration-responses to water conditioned with spermiating
males (SMW) and 3kPZS. C and D) expanded view of responses in A and B,
respectively showing response threshold concentrations. The response threshold for a
given odor is the lowest concentration that elicits a response signiﬁcantly greater than the
control (students t-test, P<0.05). The asterisks (*) denote the response threshold of
3kPZS in C and 3kPZS and SMW in D. The carrot (A) denotes the response threshold of
the other bile acids in C. Mean response magnitudes are presented as a percentage of the
response elicited by a 10'5 M L-arginine standard solution. Vertical bars represent one
standard error. Numbers by the abbreviations indicate sample sizes

60

P
m

  
   

G ’6

t- 800 - r- -

§ +PZCSA(‘(7;) €500 —<>— SMW (12)
‘9‘ . +3kPZS (12)

a 600 - +3kACA (7) .3400

T +3kPZS (7) g 300 4

1—1 400 ‘

 

         

   

 

 

 

..'1
g g 200 -
o _, en
2 20° 2 100 -
Q C
a. a.
E 0 I I I I E 0 # F I l I 1
-13 -12 -11 -10 -9 -8 -7 -6 -15 -14 -13 -12 -11 -10 -9 -8

Log molar concentration Log molar concentration

.0
o

200 ' + ACA (7) 100 —o— SMW (12)
—o— PZS (7) + 3kPZS (12)
+ 3kACA (7)
+ 3kPZS (7)

Response (% E-arg standard)
6
G

G
J

 

 

   

G
l

 

Response (% L-arg standard)
U!
6

Con -13 -12* -11 -l0" -9 Con -13 -12* -11

Log molar concentration Log molar concentration

Figure 2.

61

respectively; Figure 2D). Although the curves were similarly shaped, the SMW curve
generated slightly larger responses at equivalent 3kPZS concentrations above the
response threshold.

The responses to the L-arginine standard did not change before and during
adaptation to bile acids (Figure 3; paired t-test, P>0.10), but those to bile acids did (two-
way ANOVA, P<0.01). When used as the adapting stimuli, three of the four bile acids
completely adapted themselves (3kPZS, PZS and ACA; Figure 3; paired t-test, P<0.01,
initial responses compared to responses during adaptation; also visually compared).
Although, the response to 3kACA was signiﬁcantly lower than the initial response
(paired t-test, P<0.01), 3kACA appeared to not completely adapt itself (Figure 3B). The
analyses revealed three patterns of cross-reactivity among the four bile acids. First, when
3kPZS was used as an adapting stimulus, the response to PZS was only partially adapted,
and vice versa (Dunnett’s, P<0.05; paired t-test, P<0.01; Figure 3A and C). Second, the
responses to 3kACA and ACA were not adapted to 3kPZS (paired t-test, P>0.10; Figure
3A) and only partially adapted to PZS control levels (Dunnett’s, P<0.05; paired t-test,
P<0.03; Figure 3C). Similarly, responses to 3kPZS and PZS were not adapted to either
3kACA or ACA control levels (paired t-test, P>0.05; Figure 3B and D). Third, when
ACA was used as an adapting stimulus, the response to 3kACA was adapted to control
levels, and vice versa (Dunnett’s, P>0.05; Figure 3B and D;). When SMW was used, all

bile acid stimuli were adapted to control levels (Dunnett’s, P>0.05; Figure 3E).

62

Figure 3. Results of cross-adaptation experiments, grouped by the ﬁve adapting stimuli:
A) 3 keto-petromyzonol sulfate, 3kPZS; B) 3 keto-allocholic acid, 3kACA; C)
petromyzonol sulfate, PZS; D) allocholic acid, ACA; E) water conditioned with
spermiating males, SMW. Mean percent initial responses (PIR) are shown with
horizontal lines representing one standard error. For each adapting stimulus, the response
to self-adapted controls is underlined. Number symbols (#) signify that a particular test
stimulus was completely adapted to control levels, meaning the test stimulus PIR was not
different from the self-adapted control PIR (P>0.05, Dunnett’s test). Asterisks (*) signify
that a particular test stimulus was partially adapted, meaning the test stimulus PIR was
different from the self-adapted control PIR (P<0.05, Dunnett’s test), but the test stimulus
response during adaptation was less than the response before adaptation (responses in mV
not shown; P<0.05, paired t-test). Carrots (A) signify that a particular test stimulus was
not adapted, meaning the test stimulus response during adaptation was the same as the
response before adaptation (responses in mV not shown; P>0.05, paired t-test). Numbers
by the bars indicate sample sizes. The numbers by the abbreviations are the logarithmic
values of the molar concentrations for the tested stimuli. NT, not tested.

63

A. Adaptation to 3kPZS B. Adaptation to 3kACA

-5 L-arg -5 L-arg " 4

 
   

     
  
  
   
 

 
   
 
 
 

   
 
 

SMW SMW NT
-8 ACA -8 ACA # 3
-7 3kACA llkACA— 4 s
-9 PZS .9 P25 “ Q53
saunas -9 3kPZS A is
0 50 100 150 0 50 100 150
% initial response % initial response
C. Adaptation to P28 D. Adaptation to ACA
-5 L-arg E A 9 -5 L-arg " 5
SMW SMW NT
-8 ACA ;8_ACA 5
-7 3kACA -7 3kACA # 5
erzs .9 m A 4
-9 3kPZS * 8 -9 3kPZS " 3

     

0 50 100 150 0 50 100 150

% initial response % initial response

E. Adaptation to SMW
-5 L—arg q +14%“ 5
SM}! 3 4 :
“ml—:14 #5
-73kACA‘ NT
-9 P23 .34 #5
.9 3kPZS ‘34 #3

 

 

 

0 50 100 150
Figure 3.

64

DISCUSSION

This study establishes that the olfactory organs of adult female sea lampreys are
highly sensitive to and can electrophysiologically discriminate bile acids released by
males and larvae of the same species. Concentration-response curves showed that both
male and larval bile acids were highly stimulatory to female olfactory organs and that
there was a wide range of response dynamics associated with the four tested bile acids.

In the lower range of concentrations tested, the main component of the male pheromone
(Li et al. 2002; Yun et al. 2002), 3kPZS, with a response threshold of 10‘12 M, was 100
times more potent than the other three bile acids, whose response thresholds were 10''0
M. This difference in potency may be advantageous and critical for 3kPZS to function as
a male pheromone for two apparent reasons. First, during the spawning season, adults
and larvae occupy the same streams (Moore and Schleen 1980) in which PZS and ACA
are present at concentrations between 10'll and 10’12 M (Porkinghome et al. 2001), which
are below the detectable concentrations by female adults. Unless the real response
thresholds are lower than those determined by our EOG recording, PZS and ACA should
not interfere with female detection of 3kPZS in spawning streams. Second, sea lampreys
spawn in fast ﬂowing rivers and streams where current velocity typically reaches 1 m 3"
(Applegate 1950). Bile acids released by males would be quickly diluted by the large
volume of water passing by the male. Even though 3kPZS is produced and released at a
very high rate (ca. 0.50 mg per male per hour; Yun et al. 2002; 2003) through a highly
efﬁcient and specialized mechanism (Sieﬂces et al. 2003b), a low response threshold for

3kPZS would be advantageous for its pheromone function.

65

It is notable that in a previous study (Li et al. 1995), the olfactory epithelia of sea
lamprey migratory adults of both sexes showed EOG responses to PZS and ACA at
concentrations as low as 10'll and 10"2 M, which were lower than the 10m M determined
in this study (Figure 2A), and which were similar to the concentrations measured in
spawning streams (Porkinghome et al. 2001). The difference in measured response
thresholds is probably due to the different animal and experimental conditions between
the two studies. The animals used in Li et al. (1995) were migratory adults captured very
early in their spawning migration whereas animals used in our study were often captured
later in the migration. It has been shown that the EOG responsiveness of adult sea
lampreys to larval bile acids gradually decreases as the spawning migration progresses
(Li 1994). Furthermore, the location, water, holding tanks, odor delivery system,
digitizer, and ampliﬁer used in the study by Li et al (1995) were different from those used
in this study. Nonetheless, these two studies indicate that sea lampreys are highly
sensitive to bile acids released by individuals of the same species.

Our cross adaptation experiments demonstrated that the olfactory epithelium of
female sea lampreys electrophysiologically discriminate between different bile acids
produced by males and larvae of the same species. It appears that, from the patterns of
cross adaptation, 3kPZS and PZS represent different odors to the females. In contrast,
3kACA and ACA appear to represent the same odor or two very similar odors. Further,
the odor of 3kACA and ACA is very different from those of 3kPZS or PZS. In other
words, 3kPZS represents a unique odor among bile acids tested in this study, which may
very well be another advantageous physiological feature to promote the functionality of

3kPZS as a pheromone. The distribution of lamprey larvae in streams is not random;

66

rather, it is clumped (Applegate 1950). Thus, it is possible that at certain locations the
concentrations of PZS and ACA are higher than the average. Ovulating females, in
search of spermiating males through the 3kPZS signal (Li et al. 2002, Siefkes et al.
2003a), may be lost in locations with concentrated larvae without the ability to
discriminate 3kPZS from other conpeciﬁc bile acids.

Another issue is that females also need to discriminate bile acids released by other
aquatic and terrestrial animals. Li and Sorensen (1997) showed that PZS is
electrophysiologically discriminated by adult lamprey from all other common animal bile
acids tested. Since 3kPZS shares with PZS a combination of two molecular features (50t-
conﬁguration and a sulfate ester at carbon 24; Haslewood and Tokes 1969; Li et al.
2002), which has been found only in lamprey bile acids, it is likely that 3kPZS is also
readily discriminated from bile acids of other animals. However, the direct evidence
supporting this speculation can only come from direct testing using 3kPZS.

There were two curious phenomena that we observed during our cross adaptation
experiments. One was the apparent lack of self-adaptation when 3kACA was used as the
adapting stimulus. Contamination of the 3kACA controls may explain the lack of self-
adaptation, but this is unlikely as can be seen by the tight error bars around the self-
adapted control (Figure 3B), the complete adaptation to ACA (Figure 3D), and the purity
of the compound. Explanation may prove to be difﬁcult. The other phenomenon is the
evidence of non-reciprocal adaptation when 3kPZS and PZS are used as the adapting
stimulus. It appears that PZS as an adapting stimulus partially adapts 3kPZS by around
60%, while 3kPZS partially adapts PZS only by around 40%. Whether this pattern of

non-reciprocal adaptation is indicative of more receptor sites for PZS than 3kPZS has yet

67

to be determined. The lack of self-adaptation of 3kACA and the non-reciprocal
adaptation of 3kPZS and PZS need to be explored further. Nonetheless, the other cross
adaptation data indicate that 3kACA/ACA, 3kPZS and PZS represent different odors to
females, and support the previous hypothesis that 3kPZS functions as a pheromone (Li et
al. 2002).

Whether 3kACA could function as a component of the male pheromone remains
elusive. This compound is a potent stimulant for females (Figure 2A) and is released
only by the male lamprey during spermiation (Yun et al. 2003), exactly the same
condition when 3kPZS is released (Li et al. 2002). However, 3kACA is completely
adapted to control levels by ACA, and vice versa. It is likely a behavioral study could
conclude on its potential function as a pheromone component.

EOG data from this study also implicate that a change at carbon 3 may largely
inﬂuence the odor quality of bile acids. Several previous studies have clearly indicated
that conjugating groups (or the lack thereof) are critical in the lack of cross adaptation of
bile acids by ﬁsh olfactory epithelia. In zebraﬁsh (Danio rerio), taurine-conjugated bile
acids are more stimulatory than free or glycine-conjugated bile acids (Michel and
Lubomudrov 1995). Lake char (Salvelinus namaycush) appear to have several speciﬁc
olfactory receptor subtypes that distinguish between free, taurine-conjugated and carbon
3 sulfated bile acids (Zhang et al. 2001; Zhang and Hara 1994). Similarly, migratory
adult sea lampreys appear to electrophysiologically discriminate free, carbon 24 taurine-
conjugated, carbon 3 sulfated and carbon 24 sulfated bile acids (Li and Sorensen, 1997).
All these results are corroborated by our discovery in this study that two free bile acids

(ACA and 3kACA) are not completely cross-adapted by the two carbon 24 sulfated bile

68

acids (PZS and 3kPZS). In addition, it is noteworthy that our data clearly indicate that a
carbon 3 carbonyl or hydroxyl, neither of which is a conjugating group, also are critical
to the female lamprey olfactory epithelium in electrophysiological discrimination of
conspeciﬁc bile acids.

Our experiments using SMW have yielded mixed results. SMW is known to
induce characteristic behavioral responses in ovulating females (Li et al. 2002) and to
contain 3kPZS and 3kACA at a ratio of 25 to 1 (Li et al. 2002; Sieﬂtes et al. 2003b; Yun
et al. 2002; 2003). Corroborating with this discovery, concentration-response curves of
SMW and 3kPZS were similar in shape and magnitude (Figure 2B), especially in the
lower range of concentrations. It is likely that, at lower concentrations, the SMW
potency is largely attributable to 3kPZS. The slightly larger response magnitudes at
concentrations above 10"0 M is probably largely due to 3kACA. Presumably, SMW also
contains many other compounds that might be odorous and may contain the other bile
acids used in this study (Figure 3E). Previous chemical and electrophysiological studies
indicated that other compounds are likely to only be present in and not likely to be
stimulatory at low concentrations (Li et al. 2002; Yun et al. 2002; 2003). Further, when
collected under a condition similar to that used in this study, water from spermiating
males is stimulatory when diluted 106 times whereas water from pre-spermiating males is
stimulatory only when diluted 102 times or less (Li et al. 1994), and the main difference
between the two waters is the presence of 3kPZS and 3kACA from spermiating males (Li
et al. 2002; Yun et al. 2002).

Our cross adaptation experiments using SMW, however, do not clearly support

the notion that 3kPZS is the only stimulatory compound in diluted SMW. When SMW

69

was used as an adapting stimulus, the response to 3kPZS was adapted to control levels,
which was expected. In addition, SMW also adapted PZS and ACA to control levels,
which was not expected. SMW may contain trace amounts of PZS and ACA (if any, and
only at an amount that is less than 1% of that of 3kPZS; Yun et al. 2002), which is not
likely to be sufﬁcient to suppress responses to PZS and ACA at the concentrations used.
This issue needs to be clariﬁed in future experiments.

In conclusion, the olfactory epithelium of adult female sea lampreys is highly
sensitive to a male Speciﬁc bile acid, 3kPZS, and electrophysiologically discriminates it
from other conspeciﬁc bile acids. This provides further physiological evidence to
support the hypothesis that 3kPZS is a male pheromone (Li et al. 2002). It appears that in
addition to conjugating groups at the carbon 3 and 24 positions, other functional groups

at carbon 3 are also critical in determining the odor property of bile acids.

70

ACKNOWLEDGEMENTS

We thank Roger Bergstedt and the staff of US Geological Survey Hammond Bay
Biological Station for accommodating us in their laboratory, and Michael Twohey and
the staff of Marquette Biological Station for supplying us with sea lampreys. Dr. Sang-
Seon Yun performed ELISA analysis and helped with bile acid preparation. Members of
Li laboratory provided critique of an early version of this manuscript. The Great Lakes
Fishery Commission ﬁnanced this study. This research was approved by the Michigan
State University, All University Committee on Animal Use and Care, and complied with
all federal and state laws, policies, and rules for the humane use of laboratory animals in

research.

71

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Michigan. US Fish Wildl Serv Spec Sci Rep Fish Serv No 55

Bjerselius R, Li W, Teeter JH, Johnsen PB, Maniak PJ, Grant GC, Polkinghome CN,
Sorensen, PW (2000) Direct behavioural evidence that unique bile acids released by

larval sea lamprey (Petromyzon marinas) function as a migratory pheromone. Can J
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Doving KB, Selset R, Thommesen G (1980) Olfactory sensitivity to bile acids in
salmonid ﬁshes. Acta Physiol Scand 108: 123-131

Caprio J, Byrd RP (1984) Electrophysiological evidence for acidic, basic, and neutral
amino acid olfactory receptor sites in the catﬁsh. J Gen Physiol 842403.422

Hara TJ (1994) The diversity of chemical stimulation in ﬁsh olfaction and gustation. Rev
Fish Biol Fish 4: 1-35

Hardisty MW, Potter IC (1971) The general biology of adult lampreys. In: Hardisty MW,
Potter IC (eds) The Biology of Lampreys vol. 1, Academic Press, New York, pp. 127-
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Haslewood GA, Tokes L (1969) Comparative studies of bile salts. Bile salts of the
lamprey Petromyzon marinas L. Biochem J 1 14:179-184

Hellstrom T, Doving KB (1986) Chemoreception of taurocholate in anosmic and sham-
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Jones KA, Hara TJ (1985) Behavioural responses of ﬁshes to chemical cues: results from
a new bioassay. J Fish Biol 27:495-504

Li W (1994) The olfactory biology of sea lamprey (Petromyzon marinus). Ph. D
dissertation. University of Minnesota, St. Paul, USA

Li W, Sorensen PW, Gallaher DD (1995) The olfactory system of migratory adult sea
lamprey (Petromyzon marinas) is speciﬁcally and acutely sensitive to unique bile
acids released by conspeciﬁc larvae. J Gen Physiol 105:567-587

Li W, Sorensen PW (1997) Highly independent olfactory receptor sites for naturally
occurring bile acids in the sea lamprey, Petromyzon marinus. J Comp Physiol A
180(4):429-43 8

Li W, Scott AP, Siefkes MJ, Yan H, Liu Q, Yun S-S, Gage DA (2002) Bile acid secreted
by male sea lamprey that acts as a sex pheromone. Science 296:138-141

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Michel WC, Lubomudrov LM (1995) Speciﬁcity and sensitivity of the olfactory organ of
the zebraﬁsh, Danio rerio. J Comp Physiol A 177:191-199

Moore HH, Schleen LP (1980) Changes in spawning runs of sea lamprey (Petromyzon
marinus) in selected streams of Lake Superior after chemical control. Can J Fish
Aquat Sci 37:1851-1860

Polkinghome CN, Olson JM, Gallaher DD, Sorensen PW (2001) Larval sea lamprey
release two unique bile acids to the water at a rate sufﬁcient to produce detectable
riverine pheromone plumes. Fish Physiol Biochem 24:15-30

Siefkes MJ, Bergstedt RA, Twohey MB, Li W (2003a) Chemosterilization of male sea
lampreys does not affect sex pheromone release. Can J Fish Aquat Sci 60:23-31

Sieﬂtes MJ, Scott AP, Zielinski B, Yun S-S, Li W (2003b) Male sea lampreys,
Petromyzon marinas L., excrete a sex pheromone from gill epithelia. Biol Reprod
69:125-132.

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anguilla: a behavioral study. Environ Biol Fishes 37:197-204

Stabell OB (1987) Intraspeciﬁc pheromone discrimination and substrate marking by
Atlantic salmon parr. J Chem Ecol 13: 1625-1643

Vermeirssen ELM, Scott AP (2001) Male priming pheromone is present in bile, as well
as urine, of female rainbow trout. J Fish Biol 1039-1045

Vrieze L, Sorensen PW (2001) Laboratory assessment of the role of a larval pheromone
and natural stream odor in spawning stream localization by migratory sea lamprey
(Petromyzon marinus). Can J Fish Aquat Sci 58:2374-2385

Yun S-S, Scott AP, Li W (2003). Pheromones of the male sea lamprey, Petromyzon
marinus L: structural studies on a new compound, 3-keto allocholic acid, and 3-keto
petromyzonol sulfate. Steroids 682297-304

Yun S-S, Sieﬂces MJ, Scott AP, Li W (2002) Development and application of an ELISA
for sea lamprey male sex pheromone. Gen Comp Endocrin 129:163-170

Zhang C, Brown SB, Hara TJ (2001) Biochemical and physiological evidence that bile
acids produced and released by lake char (Salvelinus namaycush) function as
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evidenced by cross-adaptation and ligand binding assay. Chem Senses 19:579

73

CHAPTER 3

A MALE SEA LAMPREY (Petromyzon marinas) SEX PHEROMONE THAT
ATTRACTS OVULATING FEMALES IN A SPAWNING STREAM

74

ABSTRACT

The behavioral responses of female sea lampreys (Petromyzon marinus) in a
spawning stream to water conditioned with spermiating males and the synthetic bile acid,
3-keto petromyzonol sulfate were observed. Results showed that these odorants function
as a pheromone attracting only ovulating females. When released into a natural spawning
stream, ovulating, but not pre-ovulating females located and swam to the source of water
conditioned with spermiating males and also 10'‘2 Molar synthetic 3-keto petromyzonol
sulfate. Furthermore, water conditioned with spermiating males, but not synthetic 3-keto
petromyzonol sulfate induced ovulating females to remain at the site of odorant
introduction for extended periods of time. In conclusion, water conditioned with
spermiating males and synthetic 3-keto petromyzonol sulfate were able to inﬂuence
behaviors of ovulating females in a natural environment and the lowest effective

concentration of 3-keto petromyzonol sulfate that induces these behaviors is 10'12 Molar.

75

INTRODUCTION

In the Laurentian Great Lakes, the sea lamprey (Petromyzon marinas) is a high
proﬁle, highly destructive invasive species, which has caused economic and ecological
chaos in terms of their impact on the ﬁsh community (Smith and Tibbles 1980).
Although, sea lamprey management has been active for over 50 years and includes
integrated techniques such as lampricides, barriers, trapping, and sterile male releases
(Klar and Young 2002), sea lampreys continue to be a signiﬁcant source of mortality for
large and medium sized ﬁsh in the Great Lakes (Bergstedt and Schneider 1988; Kitchell
1990). For this reason, and the fact that current control techniques have the potential to
be costly and environmentally damaging (Smith and Tibbles 1980, Lamsa et al. 1980),
the development of additional means of sea lamprey management needs to occur.

At the ﬁrst Sea Lamprey International Symposium held in 1979 (Smith 1980), it
was proposed that pheromones, if identiﬁed, could be used in the management of sea
lamprey populations in the Great Lakes (Teeter 1980). One technique that could be
developed using sea lamprey pheromones is the use of pheromone-baited traps to remove
sea lampreys before they spawn, reducing their reproductive potential. Traps baited with
sex pheromones have been a common practice to control undesirable insects (Carde and
Minks 1995), but similar techniques have not been applied to vertebrate pests because
either the signals and their ﬁrnction have not been identiﬁed or where they have,
pheromones have not been shown to attract individuals of the target sex in natural

conditions or over distances great enough to be effective.

76

Recently, a sex pheromone released by spermiating male sea lamprey has been
identiﬁed and its behavioral function characterized (Li et al. 2002, and reference herein).
In the initial research, water conditioned with spermiating males (SMW) elicited
increases in swimming behaviors and ultimately attracted ovulating females in two-
choice maze behavioral assays. Chemical analyses of SMW revealed the presence of a
novel compound, 7a, 12a, 24-trihydroxy 501-cholan-3-one 24-sulfate (3-keto
petromyzonol sulfate or 3kPZS), a bile acid that is highly potent to the olfactory
epithelium of females. This bile acid, when puriﬁed also elicited the characteristic
behaviors from ovulating females in a two-choice maze. Even though the ﬁrnction of the
spermiating male sex pheromone appears to be to attract females that are ready to spawn,
behavioral tests were only conducted in a controlled laboratory-like setting (two-choice
maze), not in a natural spawning stream environment. Also, artiﬁcially synthesized or
“synthetic” 3kPZS was not used in any of the two-choice maze tests and effective
concentrations of 3kPZS were not determined.

To address whether SMW and synthetic 3kPZS are effective in a more natural
setting and if so, at what 3kPZS concentration, we examined the following speciﬁc
questions: 1) In a natural spawning stream, are ovulating females attracted to SMW; 2) In
a natural spawning stream, are ovulating females attracted to synthetic 3kPZS; and 3) If
attraction responses are found, what is the effective concentration of 3kPZS? Answering
these questions will bridge the gap between laboratory and ﬁeld behavioral experiments
and provide preliminary results on the feasibility of using pheromone-baited traps to

capture mature female sea lampreys.

77

METHODS

Collection and Maintenance of Animals

Sea lampreys were trapped or collected by hand from tributaries to Lakes Huron
and Michigan by the staff of the US. Fish and Wildlife Service, Marquette Biological
Station, Marquette, MI. All sea lampreys were transferred to the US. Geological Survey,
Hammond Bay Biological Station, Millersburg, MI. The sea lampreys were held in ﬂow-
through tanks (1000 L) with Lake Huron water at ambient temperatures. Males were
identiﬁed by a raised dorsal ridge and females by an enlarged and soft abdomen along
with no dorsal ridge (Vladykov 1949). Males and females were then placed in separate
holding tanks. Each sex was further assigned one of two maturity classiﬁcations
according to the criteria and procedure set forth by Sieﬂtes et al. (2003). For males,
individuals that did not emit sperm alter gentle pressure was applied to the abdomen were
classiﬁed as pre-spermiating males; males that did emit sperm were classiﬁed as
spermiating males. For females, individuals that did not release eggs after gentle
pressure was applied to the abdomen were classiﬁed as pre-ovulating females; females
that did release eggs were classiﬁed as ovulating females. Sea lampreys were placed in

separate tanks accordingly.

78

Behavior in a Spawning Stream

To assess whether females exhibit attraction behaviors towards SMW and
synthetic 3kPZS in a natural setting, we monitored ovulating and pre-ovulating female
locomotion in the same 65-m section of the Ocqueoc River (Figure 2) used by Li et al.
(2002). The Ocqueoc River is a historic sea lamprey spawning stream (Applegate 1950)
in which a sea lamprey barrier was recently constructed to stop adults from migrating
upstream to spawn. The absence of animals from the study area assured that there were
no background levels of odorants from other adult sea lampreys to interfere with
behavioral tests and yet offered water temperature and quality known to be suitable for
sea lamprey reproduction. At the upstream portion of the study section, an island
naturally divides the river creating two channels with approximately equal ﬂow (1.6
m3/s). A block net was placed at the downstream end of the section. An acclimation
cage (0.5 m3) for test subjects was constructed with a wood frame encased in plastic mesh
and placed at the downstream end just above the block net.

Three sets of experiments were conducted. In the ﬁrst set, females were exposed
to SMW, which were collected by holding ﬁve spermiating males in 50 L of water for 2
h. The SMW were randomly delivered to one side of the island that divides the river
(Figure 2) using a peristaltic pump at approximately 200 ml/min. Based on the release
rate of 3kPZS by spermiating males (approximately 500 ug/h/ﬁsh; Yun et al. 2002), the
ﬁnal concentration of 3kPZS in the spawning stream was approximately 2 x 10'12 M. A
blank water control was also introduced at the same rate (200 mL/min) to the channel on

the other side of the island. The second and third sets of experiments were conducted

79

 

 

A

Figure 1. The spawning stream layout used in radio tracking experiments. CI and C2
indicate points in which water conditioned with spermiating males or 3-keto
petromyzonol sulfate and control odorants were randomly introduced. A indicates the
acclimation cage in which females were held before testing. Arrows indicate water ﬂow.
The dashed line represents the downstream block net used to prevent lampreys from
exiting the study site.

80

using synthetic 3kPZS at the same concentration as measured in the conditioned water
(10’'2 M) and one order of magnitude lower (10''3 M). To mix the odorants, the
appropriate amount of synthetic 3kPZS was initially mixed with 1 ml of methanol and
then added to 50 L of water and randomly delivered to one side of the island with a
peristaltic pump at approximately 200 mL/min. A 1 ml methanol control was also mixed
with 50 L of river water and introduced at the same rate (200 ml/min) to the other side of
the island.

Tests were conducted between 0700 and 1700 hours in water temperatures
ranging from 15°C to 23°C. A day in advance of testing, four pre-ovulating or two to
four ovulating female sea lampreys (depending on availability) were ﬁtted with radio tags
designed for external mount (Advanced Telemetry System, Isanti, MN) as described by
Kelso and Gardner (2000). The tagged females and the odor sources were transported to
the study site the following morning. The tagged females were then placed in the
acclimation cage and allowed to acclimate, exposed to the two odor sources for 2 h. In
each test the females were released and their location observed visually or by a radio
receiver (Lotek Engineering Inc., Newmarket, ON, Canada) and recorded on a map grid
of the site every 5 min. If females failed to move from the release site within an hour
they were removed. If females did move from the release site they were observed until
( 1) they reached an odor source and stayed there for an hour, (2) they swam past the odor
sources, or (3) it was the end of the test (when the odorant ran out). A contingency table
was used to tally the behavior of females. Responses were categorized as swimming to
the treatment odor source, swimming to the control odor source, or not choosing an odor

source (staying at an intermediate position within the stream section or not leaving the

81

acclimation cage). A Fisher’s Exact Test was used to compare the choice of females in a
natural environment. The times in which females swam to the odor sources were also

recorded.

Independence Tests of Observed Responses

Since females were released in groups, independence of the observed responses
needed to be addressed. To accomplish this, a series of analyses were conducted to look
for evidence of differential responses among three treatments (groups of ovulating
females exposed to SMW; groups of ovulating females exposed to 10"2 M synthetic
3kPZS; individual ovulating females exposed to a group of ﬁve spermiating males [from
Li et al. 2002]). Within each treatment, each individual female was designated as an
experimental unit. The multiple response measures examined were: 1) direction of initial
movement — toward the treatment (SMW or 3kPZS), toward the control, or neither (an
intermediate position); 2) ﬁnal stopping point — treatment, control or neither and distance
from the starting point; 3) if the treatment end of the stream was reached, the average
number of time intervals (5 min. blocks) taken to get there; 4) probability of making a
forward or lateral movement — the stream was divided into 13 equal length segments with
segment 1 being the starting point and segment 13 being the end point. The stream was
divided into three channels. The outside channels were randomly assigned to the
treatment (T) or the control (C), with the center channel always being neither (N).
Females could then make 1 of 9 directional moves (CC, CN, CT, NC, NN, NT, TC, TN,

or TT) of between 0 and 12 segments in any one 5 minute period. Each trial lasted for 48

82

5-min. periods; 5) Probability of two or more females occupying the same segment x
channel space (group trials only); 6) probability of two or more females following the
same path (sequence of segment x channel spaces occupied) during the trial; 7)
probability that females tested as a group exhibited different movement patterns (path

followed) than did females tested individually.

83

RESULTS

Water conditioned with spermiating males and synthetic 3kPZS inﬂuenced the
behavior of ovulating females within the spawning stream section (Table 1). The
behavior of all female sea lampreys in the Ocqueoc River varied greatly, ranging from
fast movement upstream towards one of the odor sources to no movement from the
acclimation cage. Females were not biased to either side of the stream (data not shown),
but their movement was largely inﬂuenced by the odorants. When exposed to SMW,
seven of ten ovulating females swam to and stayed for extended periods of time (2 h) at
the exact point of odorant introduction repeatedly placing their nostril directly under the
tube, while three did not choose an odor source. Of the seven that did choose, the mean
time to swim the 65 m to the odorant was 29 min. No ovulating females swam to the
control odorant. Among the ten pre-ovulating females exposed to SMW, one swam to
the point of odorant introduction, none swam to the control odorant, and nine did not
choose an odor source. When exposed to 10''2 M 3kPZS, seven of ten ovulating females
swam to, paused, and swam past the point of odorant introduction, one swam to the
control odorant, and two did not choose an odor source. Of the seven that did choose, the
mean time to swim the 65 m to the odorant was 94 min. Among the ten pre-ovulating
females exposed to 10''2 M 3kPZS, one swam to, paused, and swam past the point of
odorant introduction, one swam to the control odorant, and eight did not choose an odor
source. All ten ovulating females tested with 10''3 M 3kPZS did not choose an odor
source. The distributions of choices differed signiﬁcantly between ovulating females and

pre-ovulating females when both SMW and 10'12 M 3kPZS were used as odor sources

84

Table 1. The distribution of ovulating and pre-ovulating female sea lampreys swimming
to the treatment (TRT), control (CON), or staying at an intermediate position (INT)
within a section of a known sea lamprey spawning stream when water conditioned with
spermiating males (SMW) and 10"2 M 3-keto petromyzonol sulfate (synthetic 3kPZS)
treatments were used. The distributions of choices were signiﬁcantly different between
ovulating and pre-ovulating females for each treatment suggesting an attraction by
ovulating females to the treatments (Fisher’s Exact Test, 2-Tail, P < 0.03).

 

 

SMW Synthetic 3kPZS
Choice Choice
Test TRT CON INT SYN CON TNT
Ovulating female 7 0 3 7 l 2
Pre-ovulating female 1 0 9 l - l 8

 

85

(Fisher’s exact test, P<0.03). Further, ovulating females remained around the point of
odorant introduction when SMW were used, but not when 10‘[2 M 3kPZS was used,
however, this searching behavior was not quantiﬁed.

Independence analyses demonstrated that the females tested as a group did not
respond differently from those tested individually. The analyses strongly showed that
3kPZS is an effective attractant. There was no difference detected in the direction of
initial movement among the three trials (x2 = 1.53, d.f. = 4, p > 0.05). Females tested
within a group (SMW or 3kPZS) were as likely to remain in the center channel or move
to the treatment or control channel as were females tested individually (individual tests
from Li et al. [2002]). Seventy percent (7 of 10) of the females tested in the group 3kPZS
trial, 70% (7 of 10) of those tested in the group SMW trial, and 67% (8 of 12) of those
tested individually (with ﬁve spermiating males as the odor source) reached the end of
the treatment channel (segment 13) during the trial period. The average number of time
periods required to reach the end of the treatment channel was 4.5 (range = 2 - 9) for the
individually tested females, 5.7 (range = 1 - 11) for the group of SMW tested females and
18.7 (range = 5 - 36) for the group of 3kPZS tested females. The results for the group
3kPZS test are inﬂated by one trial in which 3 of the 4 females reached the end of the
treatment channel, but took 29, 34, and 36 time periods to do so. If these 3 individuals
are removed from the analysis, the average number of time periods for the group 3kPZS
tested females drops to 8.0 (range = 5 - 15). If lampreys being tested in a group are
moving in a correlated manner, we should expect that the average time to reach the end
of the treatment channel would be lower than that observed for lampreys that were tested

independently. The opposite was true.

86

The movements of females were classiﬁed into one of three categories: within a
channel, toward the treatment channel or away from the treatment channel. Within each
of the three trials, because most of the females ended up in the end segment of the
treatment channel, the highest probability for movement was within that channel. The
probability of movement within the treatment channel was 0.60 in the group 3kPZS trial,
0.64 in the group SMW trial, and 0.71 in the individual trial. The probability of moving
toward the treatment channel was the same in each of the three trials. The probability of
moving away from the treatment channel was 0.01 in the group trials and 0.002 in the

individual trials.

87

DISCUSSION

Results show that odorants released by spermiating males signiﬁcantly inﬂuenced
the behavior of ovulating females and not pre-ovulating females in a natural spawning
stream environment. Using SMW, seven of ten ovulating females swam the 65 m
directly to and stayed at the point of odorant introduction. In addition, using 10'12 M
3kPZS, seven of ten ovulating females swam the 65 m to the point of odorant
introduction, paused, and swam upstream, not staying at the point of odorant
introduction. Finally, pre-ovulating females did not move upstream when exposed to the
above odorants.

Results also indicate that the spermiating male pheromone is active at extremely
low concentrations. After SMW were found to induce attraction and search responses
from ovulating females, they were analyzed for 3kPZS concentration using ELISA (Yun
et al. 2002). The SMW were found to contain approximately 2 x 10'12 M 3kPZS. In
comparison, 10'12 M synthetic 3kPZS was also able to induce an attraction response from
ovulating females, but 10'” M synthetic 3kPZS was not able to induce the attraction
response. Based on these data, it is logical to conclude that the lowest effective
concentration of 3kPZS is approximately 10‘12 M. An effective low concentration makes
sense in the context of sea lamprey biology. Sea lampreys spawn in fast ﬂowing water
where current velocity reaches 1 m/s (Applegate 1950). The passing water would quickly
dilute the 3kPZS released by males. Even though 3kPZS is produced and released at a

very high rate (around 0.50 mg/male/h; Yun et al. 2002, 2003) and through a highly

88

a.“ ﬁn.

efﬁcient and specialized mechanism (Sieﬂ<es et al. 2003b), a low effective concentration
would be advantageous for its pheromone function in the stream environment.

Results further justify the pursuit of incorporating pheromone-baited traps into
integrated sea lamprey management. Past research by Li et al. (2002), has shown that
3kPZS, a bile acid uniquely released by spermiating males, increases search behavior in
ovulating females and ultimately attracts them in a two-choice maze, but behavioral
function was not demonstrated in a natural environment. The current research addresses
this gap in knowledge and moves these experiments to the ﬁeld, demonstrating the
effectiveness of low concentrations of 3kPZS (10’12 M) over a distance of 65 m. Another
important result was that SMW were able to lure and keep ovulating females at the exact
point of SMW introduction. This preliminary research sets the stage for the development
of a pheromone-baited trapping technique for integrated sea lamprey management.

Overall design restrictions precluded any direct test for independence of the
observed responses of ovulating females tested in groups. The cost associated with
obtaining synthetic 3kPZS, the availability of ovulating females, and the physical
limitations of the stream section made it impractical to test ovulating females
individually. The only reasonable design necessitated testing two to four females each
trial. Additionally, only three trials using synthetic 3kPZS and three trials using SMW
could be conducted. Ironically, if the synthetic compound had been ineffective,
independence would have been easier to document, because the response pattern would
have been random. The best evidence that ovulating females tested in groups tend to act
independently comes from the ﬁnal three analyses: 1) there were no recorded cases of

two or more ovulating females occupying the same segment x channel space (except after

89

they reached the end of a channel), as would be expected if they were moving in concert,
2) there were no recorded cases of ovulating females tested in groups following the same
path during the trial, meaning within each group, each ovulating female followed a
unique path as it moved to the end of the treatment channel and each took a different
number of time periods to reach the end of the treatment channel, and 3) the paths
followed by females tested in a groups mimic those followed by females tested
individually (from Li et al. 2002).

Although an attraction response was seen using 10'12 M 3kPZS, this
concentration, unlike SMW, did not keep ovulating females around the site of odorant
introduction for extended periods of time. This result is puzzling because of the ability of
puriﬁed 3kPZS from SMW (theoretically the same as synthetic 3kPZS) to keep ovulating
females in close proximity of its release site (Li et al. 2002). Keeping ovulating females
at nest sites is likely an important pheromonal mechanism precluding reproduction.

One reason for the inability of synthetic 3kPZS to keep ovulating females in close
proximity could be the composition of the male pheromone. In insects, precise
concentrations of pheromone components are needed to induce attractions from
conspeciﬁcs (Vickers et al. 1991). It is possible that in addition to 3kPZS, there may be
other unidentiﬁed components of the pheromone that are needed to elicit the full
behavioral response from ovulating females. These compounds could also be present in
puriﬁed 3kPZS samples. More research is needed to further characterize the behavioral
function of 3kPZS, ﬁnd the exact concentrations of 3kPZS released by spermiating

males, and to explore the possibility of additional components of the male pheromone.

90

Nevertheless, results demonstrate that synthetic 3kPZS is able to attract ovulating females
in a natural stream environment.

In summary, in a spawning stream SMW and synthetic 3kPZS were able to attract
ovulating females in a natural environment. The lowest effective concentration of 3kPZS
appeared to be 10‘'2 M, which makes sense in the context of sea lamprey ecology.
Furthermore, SMW, but not synthetic 3kPZS were able to keep ovulating females at the
site of odorant introduction. It is evident that 3kPZS is part of a mixture that is the
spermiating male pheromone and that there may be other compounds that have yet to be
identiﬁed. More research is needed to further deﬁne the ﬁmction of 3kPZS and also to

determine if there are other components of the spermiating male pheromone.

91

ACKNOWLEDGEMENTS

I would like to thank the staffs of Hammond Bay Biological Station and
Marquette Biological Station for providing research facilities, equipment and sea
lampreys for this study. I would also like to thank Dr. Scott Winterstein for his help with
independence tests and John Kelso and the Department of Fisheries and Oceans, Ontario,
Canada for the use of their telemetry equipment. Lastly, a special thanks to Dolly Trump
and Lydia Lorenz who provided access to their private land during this study. This study

was supported by the Great Lakes Fishery Commission.

92

REFERENCES

Applegate, V.C. 1950. Natural history of the sea lamprey (Petromyzon marinus) in
Michigan. United States Fish and Wildlife Service Special Scientiﬁc Report. Fisheries
Service, 55, 237 pp.

Bergstedt, R.A., and GP. Schneider. 1988. Assessment of sea lamprey (Petromyzon
marinas) predation by recovery of dead lake trout (Salvelinus namaycush) from Lake
Ontario. Canadian Journal of Fisheries and Aquatic Sciences, 45(8), 1406-1410.

Carde, RT, and AK. Minks. 1995. Control of moth pests by mating disruption: success
and constraints. Annual Review of Entomology, 40, 559-85.

Kelso, J .R.M., and W.M. Gardner. 2000. Emigration, upstream movement, and habitat
use by sterile and fertile sea lampreys in three Lake Superior tributaries. North
American Journal of Fisheries Management, 20, 144-153.

Kitchell, J .F. 1990. The scope for mortality caused by sea lamprey. Transaction of the
American Fisheries Society, 119, 642-648.

Klar, GT, and R.J. Young. 2002. Integrated management of sea lampreys in the Great
Lakes 2002. USFWS, Department of Fisheries and Oceans Canada, Annual Report to
the Great Lakes Fishery Commission, Marquette, Michigan.

Lamsa, A.K., C.M. Rovainen, D.P. Kolenosky, and L.H. Hansen. 1980. Sea lamprey
control — where to from here? Report of the SLIS control theory task force. Canadian
Journal of Fisheries and Aquatic Sciences, 37(11), 2175-2192.

Li, W., A.P. Scott, M.J. Sieﬂtes, H. Yan, Q. Liu, S.-S. Yun, and DA. Gage. 2002. Bile
acid secreted by male sea lamprey that acts as a sex pheromone. Science (Washington
DC), 296, 138-141.

Siefkes, M.J., R.A. Bergstedt, M.B. Twohey, W. Li. 2003a. Chemosterilization of male
sea lampreys (Petromyzon marinas) does not affect sex pheromone release. Canadian
Journal of Fisheries and Aquatic Sciences, 60, 23-31.

Sieﬂtes, M.J., A.P. Scott, B. Zielinski, S.-S. Yun, and W. Li. 2003b. Male sea lampreys,
Petromyzon marinus L., excrete a sex pheromone from gill epithelia. Biology of
Reproduction, 69, 125- 132.

Smith, BR. 1980. Introduction to the proceedings of the 1979 sea lamprey international
symposium (SLIS). Canadian Journal of Fisheries and Aquatic Sciences, 37, 1585-
1587.

93

Smith, BR, and J .J . Tibbles. 1980. Sea lamprey (Petromyzon marinus) in Lakes Huron,
Michigan, and Superior: History of invasion and control, 1936-78. Canadian Journal
of Fisheries and Aquatic Sciences, 37, 1780-1801.

Teeter, J. 1980. Pheromone communication in sea lampreys (Petromyzon marinus):
implications for population management. Canadian Journal of Fisheries and Aquatic
Sciences, 37, 2123-2132.

Vickers, N.J., T.A. Christensen, H. Mustaparta, and TC. Baker. 1991. Chemical
communication in heliothine moths. 111. Flight behavior of male Helicoverpa zea and
Heliothis virescens in response to varying ratios of intra- and interspeciﬁc sex
pheromone components. Journal of Comparative Physiology A, 169, 275-280.

Vladykov, V.D. 1949. Quebec lampreys. 1.-List of species and their economical
importance. Department of Fisheries, Province of Quebec, Contrib. 26, 67 pp.

Yun, S.-S., A.P. Scott, and W. Li. 2003. Pheromones of the male sea lamprey,
Petromyzon marinas L.: structural studies on a new compound, 3-keto allocholic acid,
and 3-keto petromyzonol sulfate. Steroids, 68, 297-304.

Yun, S.-S., M.J. Sieﬂces, A.P. Scott, and W. Li. 2002. Development and application of an

ELISA for a sex pheromone released by male sea lampreys (Petromyzon marinas L.).
General and Comparative Endocrinology, 129, 163-170.

94

CHAPTER4

C HEMOSTERILIZATION OF MALE SEA LAMPREYS (Petromyzon marinas)
DOES NOT AFFECT SEX PHEROMONE RELEASE

Sieﬂces, M. J., Bergstedt, R. A., Twohey, M. B., and Li, W. 2003. Chemosterilization of

male sea lampreys (Petromyzon marinas) does not affect sex pheromone release.
Canadian Journal of Fisheries and Aquatic Sciences, 60:23-31.

95

ABSTRACT

Release of males sterilized by injection with bisazir is an important experimental
technique in management of sea lamprey (Petromyzon marinus), an invasive, nuisance
species in the Laurentian Great Lakes. Sea lampreys are semelparous and sterilization
can theoretically eliminate a male’s reproductive capacity and, if the ability to obtain
mates is not affected, waste the sex products of females spawning with him. It has been
demonstrated that spermiating males release a sex pheromone that attracts ovulating
females. We demonstrated that sterilized, spermiating males also released the pheromone
and attracted ovulating females. In a two-choice maze, ovulating females increased
searching behavior and spent more time in the side of the maze containing chemical
stimuli from sterilized, spermiating males. This attraction response was also observed in
spawning stream experiments. Also, electro-olfactograms showed that female olfactory
organs were equally sensitive to chemical stimuli from sterilized and nonsterilized,
spermiating males. Finally, fast atom bombardment mass spectrometry showed that
extracts from water conditioned with sterilized and nonsterilized, spermiating males
contained the same pheromonal molecule at similar levels. We concluded that injection

of bisazir did not affect the efﬁcacy of sex pheromone in sterilized males.

96

INTRODUCTION

The sea lamprey (Petromyzon marinus) is a nonindigenous, nuisance species in
the Laurentian Great Lakes and a potentially lethal parasite feeding on the blood of larger
ﬁshes such as lake trout (Salvelinus namaycush) and lake Whiteﬁsh (Coregonus
clupeaformis). Its establishment in the upper Great Lakes during the 19203 through
19405 was a signiﬁcant factor in the collapse of commercial ﬁsheries and brought about
dramatic biological and ecological changes in the ﬁsh communities (e.g., Smith 1971;
Coble et al. 1990). Despite an intensive program started in the 19505 to control their
populations (Smith and Tibbles 1980), the sea lamprey remains a factor in the mortality
of larger ﬁshes in the Great Lakes (e.g., Spangler et al. 1980; Kitchell 1990).

An important new method being experimentally implemented in sea lamprey
management is the sterile male release technique (e. g., Bergstedt et al. 2003; Schleen et
al. 2003; Twohey et al. 2003a). In this technique, the chemosterilant bisazir (P,P-bis(1-
azirindinyl)-N-methylphosphinothioic amide) is injected intraperitoneally into adult
males (Hanson and Manion 1978). Sperm from males injected with bisazir can still
fertilize eggs, but the resulting embryos die before reaching the larval stage (Hanson and
Manion 1980). Laboratory and ﬁeld studies with bisazir treatment demonstrated that
sterilized males mate successfully with females (Hanson and Manion 1978) and when
released into spawning streams reduce the abundance of sea lamprey offspring (Bergstedt
et al. 2003). In the current application of the sterile male release technique, the observed
proportion of sterilized males in male spawning populations was typically near expected

values calculated by mark and recapture estimates, but the overall percent egg viability

97

was consistently higher than the theoretical effect of the number of sterilized males
released (Bergstedt et al. 2003). One factor potentially contributing to the difference
between the actual and theoretical egg viability could be the effect of bisazir on
pheromone production of male sea lampreys. Nonsterilized, spermiating male sea
lampreys release a potent sex pheromone, 7a, 12a, 24-trihydroxy 5a-cholan-3-one 24-
sulfate, which induces preference and locomotor activities of ovulating females (Li et al.
2002). This pheromone functions to attract females that are ready to spawn. Clearly, if
bisazir affected the release of this pheromone, the ability of sterilized males to attract
mates would be reduced. The present study explores this possibility.

As invasive ﬁsh species increasingly threaten the health of our aquatic
ecosystems, the sterile male release technique is one of the few potential control methods
with no identiﬁable environmental effects. It has the potential to become an important
technique in management of these nuisance species, for which bisazir could be used as a
sterilant. Where studied, most ﬁsh species are shown to rely heavily on the olfactory
sense to mediate reproduction (e.g., Solomon 1977; Liley 1982; Stacey et al. 1994).
Although each species would have to be tested independently, initial determination of
whether sterilization of sea lampreys with bisazir affects sex pheromone biosynthesis and
release would bear strongly on its potential for broader use.

The overall goal of this study was to determine if bisazir-sterilized, spermiating
male sea lampreys release the same sex pheromone at similar levels found in
nonsterilized, Spermiating males and if they elicit the same behavioral response from
adult sea lampreys. We applied a multidisciplinary approach to characterize whole

animal and electrophysiological responses to, as well as chemistry of, chemical stimuli

98

produced by sterilized males. The following speciﬁc questions were posed. (i) In a two-
choice maze, do ovulating females show preference and searching behaviors when
exposed to water conditioned with bisazir-sterilized, spermiating males? (ii) In a
spawning stream, are female sea lampreys attracted to water conditioned with bisazir-
sterilized, spermiating males? (iii) Do female olfactory organs show the same
electrophysiological response to water conditioned with bisazir-sterilized and
nonsterilized, spermiating males? (iv) Do extracts of water conditioned with bisazir-
sterilized and nonsterilized, spermiating males contain the same pheromone molecule?
Our results showed that bisazir-sterilized, spermiating male sea lampreys released the

same sex pheromone at similar levels found in nonsterilized, spermiating males.

99

MATERIALS AND METHODS

Collection and maintenance of animals

Adult sea lampreys were collected from tributaries to lakes Huron and Michigan
by the staff of the U. S. Fish and Wildlife Service, Marquette Biological Station,
Marquette, Mich. The animals were transported to the sterilization facility and main
laboratory at the U. S. Geological Survey, Hammond Bay Biological Station,
Millersburg, Mich. Males were identiﬁed by their raised dorsal ridge and females by
their enlarged, soft abdomen and absence of dorsal ridge (Vladykov 1949). Males
averaged 470 mm and 233 g while females averaged 465 mm and 240 g. The sexes were

separated and held in ﬂow-through tanks (1000 L) with Lake Huron water (7 °C to 20

°C).

Sterilization of male sea lamprey

Males were sterilized in the sterilization facility by robotic injection with a dose
of 100 mg-kg body weight'1 of bisazir (Hanson and Manion 1978; 1980; Twohey et al.
2003a). Quality assurance monitoring showed that males used during this study received
no less than the correct dose of bisazir (Twohey et al. 2003a). Sterilized males were held
in the sterilization facility for at least 48 h after injection before being used in any

procedure.

100

Identification of maturity

Each sex was further assigned one of two maturity classiﬁcations. For males,
gentle pressure was applied to the abdomen to induce emissions into a petri dish. White
emissions indicated spermiation. Depending on treatment status, males that did not emit
sperm were classiﬁed as nonsterilized, prespermiating males or as sterilized,
prespermiating males. Those that did emit sperm were classiﬁed as nonsterilized,
spermiating males or as sterilized, spermiating males. To determine whether white
emissions correctly indicated spermiation, emissions from 10 males classiﬁed as
spermiating and 10 classiﬁed as nonspermiating were checked under a microscope for
sperm. All 20 males were classiﬁed correctly. For females, gentle pressure was applied
to the abdomen to induce egg emission. Females that did not release eggs were classiﬁed
as preovulating females and those that did release eggs were classiﬁed as ovulating

females.

Induction of spermiation and ovulation

Sterilized and nonsterilized males were allowed to spermiate naturally or were
induced to spermiate by holding them at 18 °C and then injecting them intraperitoneally
with [D-Ala6]-luteinizing hormone-releasing hormone (pGlu-His-Trp-Ser—Tyr-D-Ala-
Leu-Arg-Pro-Gly-NHZ, Sigma Chemical Co., St. Louis, M0.) at a dose of 100 pg-kg body
weight'l (Li 1994). This injection was repeated 3 and 5 days later. Males were checked

each day until spermiation occurred. Females were treated similarly to induce ovulation.

101

Chemical stimuli used in experiments

Either male sea lampreys or water conditioned with male sea lampreys were used
as the chemical stimuli in experiments. Conditioned water was collected by holding
individual males of classiﬁcation for 4 h in polyethylene buckets ﬁlled with 10 L of
water. These buckets were aerated and kept in a water bath of 18 °C. The conditioned
water was either (i) used immediately for behavioral assays, (ii) frozen immediately at
-80 °C for subsequent electro-olfactographic analyses, or (iii) extracted using C-18 solid-

phase extraction cartridges and the elute frozen at -80 °C for chemical analysis.

Experiment 1: In a two-choice maze, do ovulating females show preference and
searching behaviors when exposed to water conditioned with bisazir-sterilized,
spermiating males?

Preference and searching tests were used to assess the attraction behaviors of
adult sea lampreys of each sex and maturity class to chemical stimuli produced by
sterilized males of both maturity classes. Preference behaviors were measured as the
amount of time spent in each side of the maze, and searching behaviors were measured as
the amount of time spent swimming at the head of each side of the maze and were
categorized as described by Li et al. (2002). These tests were carried out in the same
two-choice maze, under similar conditions and following the same protocol of Li et al.
(2002). Brieﬂy, in each test, the behavior of a single test subject was video recorded
before and alter the introduction of chemical stimuli from either sterilized, spermiating or

prespermiating males into the odor chamber of the side of the maze chosen randomly by

102

coin toss. The chemical stimuli were either ﬁve sterilized males per test (placed directly
into the odor chamber) or water conditioned with sterilized, spermiating males (delivered
into the odor chamber at 75 mL-min'I using a peristaltic pump). Tests were conducted
between 0700 and 1700 in water temperatures that ranged from 12 °C to 24 °C.

The times spent in each side of the maze and the time spent swimming at the head
of each arm of the maze by the test animal in the treatment (stimulus) and control (no
stimulus) side before and after chemical stimulus introduction were summed from the
videotapes by a reviewer unaware of which side contained the stimulus. The data were
used to calculate an index of preference or searching (I), described by Li et al. (2002) for
each individual test subject: I = (At/(At+Bt)) — (Ac/(Ac+Bc)), where Bt and At are the
time spent or time spent searching in the treatment side of the maze before and aﬁer
chemical stimulus introduction, respectively, and Bc and Ac are the time spent or time
spent searching in the control side of the maze before and after chemical stimulus
introduction, respectively. These indices compare test subject behavior in each side of
the maze before and after chemical stimulus introduction. A positive I value indicates a
preference or increase in preference or searching behavior, while a negative value
indicates an avoidance or decrease in the behaviors.

The I values were analyzed with a two-tailed Wilcoxon Signed Ranks Test (Rao
1998) to determine if there was differences in preference and searching behavior in each
combination of test subject and chemical stimulus source class. Sample sizes varied due
to availability of test subjects (Tables 1 and 2). Tests in which proportions and I values
could not be generated were not used in analyses. These situations comprised

approximately 20% of all tests and occurred when test subjects did not explore both sides

103

of the maze in the initial recording period or did not explore either side of the maze in the
experimental recording period, causing zero index values dictated by the Wilcoxon

signed rank test to be deleted.

Experiment 2: In a spawning stream, are female sea lampreys attracted to water
conditioned with bisazir-sterilized, spermiating males?

To assess whether sterilized, spermiating male chemical stimuli attract females in
their spawning habitat, we monitored ovulating and preovulating female responses to
sterilized male chemical stimuli under similar conditions and in the same 65-m section of
the Ocqueoc River used by Li et al. (2002). Tests were conducted between 0700 and
1700 in water temperatures ranging from 12 °C to 24 °C using the protocol of Li et al.
(2002). A day before testing, a female sea lamprey (ovulating or preovulating) was ﬁtted
externally with a radio tag (Model 393, Advanced Telemetry System, Isanti, Minn.)
according to Kelso and Gardner (2000). The tagged female and the ﬁve sterilized,
spermiating and ﬁve sterilized, prespermiating males were transported to the study site on
the following morning. The sterilized, spermiating and sterilized, prespermiating males
were randomly assigned to two upstream cages. The tagged female was placed in a
downstream acclimation cage, exposed to the two chemical stimuli sources for 2 h, and
then released.

In each test, the subject’s location was observed visually or determined with a
directional radio antenna and receiver (Lotek Engineering Inc., Newmarket, Ont.) and
recorded on a map grid of the site every 5 min. If test subjects failed to move from the

release site within 1 h, they were removed. If test subjects did move from the release site,

104

they were observed until (1) they reached one cage or the other and stayed there for l h;
(ii) they swam past the cages; or (iii) 4 h elapsed from the start of the test. A contingency
table was used to tally the behavior of ovulating (N = 15) and preovulating female (N =
10) test subjects. Responses were categorized as swimming to the sterilized, spermiating
males, swimming to the sterilized, prespermiating males, or not choosing (staying at an
intermediate position within the stream section or not leaving the acclimation cage). A
Fisher’s Exact Test was used to compare the choice of females (PROC FREQ; SAS

Institute Inc., Cary, NC.)

Experiment 3: Do female olfactory organs show the same electrophysiological
response to water conditioned with bisazir-sterilized and nonsterilized, spermiating
males?

The olfactory potency of an L-arginine standard and chemical stimuli from
sterilized, spermiating males, nonsterilized, spermiating males and prespermiating males
were examined with electro-olfactogram (EOG) recordings of female sea lampreys. EOG
recordings measure summated generator potentials of the olfactory neurons (Ottoson
1956; Getchell 1974). The recordings were conducted according to the procedures
established by Li et al. (1995). Brieﬂy, a female was anaesthetized with metomidate
hydrochloride (3 mg-kg'l body weight) (Syndel, Vancouver, BC), immobilized with
gallamine triethiodide (150 mg-kg’l body weight) (Sigma Chemical Co., St. Louis, Mo.),
and placed in a water-ﬁlled trough. The head of the female remained above the water
and the gills were supplied with aerated Lake Huron water. The olfactory lamellae were

then exposed and perfused with water from a source that could deliver either clean water

105

or the same clean water containing chemical stimuli on demand. Differential electrical
responses between the skin surface and the sensory epithelia in response to each chemical
stimuli were recorded using two Ag/AgCl electrodes (type EH-lS, World Precision
Instruments, Sarasota, Fla.) ﬁlled with 3 M potassium chloride and bridged with glass
capillary tubes ﬁlled with 8% gelatin - 0.9% saline. Two experiments were conducted
using this setup.

In the ﬁrst experiment, we determined dose-response relationships (Li et al. 1995)
for water conditioned with sterilized, spermiating (N = 8), nonsterilized, spermiating (N =
8) and nonsterilized, prespermiating (N = 4) males as test stimuli. In a given trial, a 10‘5
M L-arginine standard was pulsed into the olfactory epithelium of a female for 5 s and
the EOG response measured to establish a baseline of electrical activity. Next, blank
control water was introduced and the response measured to conﬁrm the absence of
response from the clean water supply used both to perfuse the olfactory organ and to
dilute the chemical stimuli for testing. Increasing concentrations of test stimuli starting at
106 times dilution were then introduced and the responses measured. Measuring the
response to the L-arginine standard and blank controls again at the end of the dilution
series concluded each trial. The epithelium of each female was allowed to recover at
least 3 min between stimuli, and each of the test stimuli concentrations were assayed at
least twice on a female. Female EOG response magnitudes were measured in mV and
expressed as a percentage of the L-arginine standard.

In the second experiment, we used cross-adaptation as described by Li and
Sorensen (1997) to determine the olfactory sensitivity of females to water conditioned

with sterilized (N = 5) and nonsterilized (N = 5), spermiating males. In a given trial,

106

baseline olfactory responses of females to blank control water, a L-arginine standard, and
the test stimuli (water conditioned with sterilized and nonsterilized, spermiating males)
were recorded using EOG. The test stimuli were used at concentrations that elicited
equipotent olfactory responses at about 200% of the L-arginine standard. During
adaptation, the olfactory epithelium of the female was continually perfused with the
adapting stimulus (either sterilized or nonsterilized, spermiating male test stimuli) for 5
min after which a 5-s pulse of the same stimulus at the same concentration being used for
the adaptation was introduced to the nose to establish that the sensory epithelium was
completely adapted. Next, a 5-s pulse of the L-arginine standard was introduced to
conﬁrm that the olfactory epithelium of the female was still active. A 5-s pulse of the
alternate test stimulus (not being used as the adapting stimulus) was then introduced to
see if any responses were generated. Switching the epithelial adapting stimulus back to
clean water completed the trial. The epithelium of the female was allowed to recover for
30 min and then it was tested again using the alternate test stimulus as the adapting
stimulus. The effects of adaptation on female responses to male stimuli were analyzed
with a two-way analysis of variance (ANOVA). Once the main effect of adaptation had
been established, effects of each adapting stimuli on olfactory responsiveness to other
stimuli were determined by Student’s t tests with equal variance (PROC GLM, SAS

Institute Inc., Cary, NC.)

107

Experiment 4: Do extracts of water conditioned with bisazir-sterilized and
nonsterilized, spermiating males contain the same pheromone molecule?

Water conditioned with sterilized (N = 3) and nonsterilized (N = 3), spermiating
males were separately pre-ﬁltered with No. 3 Whatman ﬁlter paper, passed through
methanol-activated C-18 solid phase extraction cartridges (Waters, Milford, Mass.) and
eluted with 100% methanol. All six extracts were assayed for relative abundances of
molecules by subjecting them to fast atom bombardment mass spectrometry (10 kV)

analyses at both negative and positive ionization modes.

108

RESULTS

The total time spent and time spent searching by test subjects in the two sides of
the maze before chemical stimulus introduction did not differ signiﬁcantly (P > 0.10; data
not shown), indicating that adults were not biased to either side. Ovulating females,
however, showed strong preference responses to sterilized, spermiating male chemical
stimuli (Table 1). Eleven out of 12 ovulating females spent more time in the side of the
maze containing ﬁve sterilized, spermiating males (P < 0.01, N = 12), and 9 of 10
ovulating females spent more time in the side containing water conditioned with
sterilized, spermiating males (P < 0.02, N = 10). In contrast, none of the three other
classes of test subjects showed a preference response to sterilized, spermiating male
chemical stimuli (P > 0.10, see Table 1 for sample sizes), and ovulating females did not
Show a preference response to sterilized, prespermiating male chemical stimuli (P > 0.10,
N = 10; data not shown).

Consistent with the preference results, there was an increase in searching behavior
by ovulating females exposed to chemical stimuli from sterilized, spermiating males
(Table 2): all eight ovulating females spent signiﬁcantly more time searching in the side
of the maze containing ﬁve sterilized, spermiating males (P < 0.01, N = 8) and seven of
eight ovulating females spent signiﬁcantly more time searching in the side containing
water conditioned with sterilized, spermiating males (P < 0.01, N = 8). In contrast, none
of the three other classes of test subjects showed signiﬁcant increases or decreases in

searching behavior (P > 0.10, see Table 2 for sample sizes), and ovulating females did not

109

Table 1. Chemical stimuli from sterilized, spermiating male sea lampreys (Petromyzon
marinus) induced preference responses from ovulating females only. N: sample size.
Preference is the number of test subjects that spent proportionately more time after
stimulus introduction in treatment (scented) side. P-values were determined using a
Wilcoxon Signed Ranks Test (2-tailed) using indices of preference. NS: not signiﬁcant
(P > 0.10). Preference ratio is the mean ratio (standard deviation) of the time spent in
seconds in the treatment/control sides after stimulus introduction. *indicates ovulating
female tests using water conditioned with sterilized, spermiating males as the stimulus

SOUI'CC.

 

 

Test subject N Preference P Preference ratio
Ovulating female 12 11 < 0.01 7.0 (5.5)
*Ovulating female 10 9 < 0.02 2.0 (1.0)
Pre-ovulating female 10 6 NS 1.7 (1.0)
Non-sterilized 11 7 NS 1.4 (1.8)
spermiating male

Non-sterilized 10 6 NS 2.2 (1.6)

pre-spermiating male

 

110

Table 2. Chemical stimuli from sterilized, spermiating male sea lampreys (Petromyzon
marinus) increased searching responses from ovulating females only. N: sample size.
Searching is the number of test subjects that spent proportionately more time swimming
after stimulus introduction in treatment (scented) side. P-values were determined using a
Wilcoxon Signed Ranks Test (2-tailed) using indices of preference. NS: not signiﬁcant
(P > 0.10). Searching ratio is the mean ratio (standard deviation) of the time in seconds
spent swimming in the treatment/control sides after stimulus introduction. *indicates
ovulating female tests using water conditioned with sterilized, spermiating males as the
stimulus source.

 

 

Test subject N Searching P Searching ratio
Ovulating female 8 8 < 0.01 3.9 (1.5)
*Ovulating female 8 7 < 0.01 3.3 (4.0)
Pre-ovulating female 7 3 NS 1.3 (1.3)
Non-sterilized 10 5 NS 0.8 (1 .2)

spermiating male

Non-sterilized 8 5 NS 2.4 (2.7)
pre-spermiatinﬁg male

 

lll

show signiﬁcant increases or decreases in searching behavior (P > 0.10, N = 8; data not
shown) in response to sterilized, prespermiating male chemical stimuli.

The chemical stimuli released by sterilized, spermiating males inﬂuenced the
behavior of ovulating females, but not of preovulating females, within the spawning
stream section. Females were not biased toward either side of the stream. Among the 15
ovulating females tested, 10 swam to and then stayed at the cage containing sterilized,
spermiating males and ﬁve did not choose a stimulus source. Of the ten that did choose,
the mean time to swim the 65 m to the sterilized, spermiating males was 29 min. No
ovulating females swam to the cage containing sterilized, prespermiating males. Among
the 10 preovulating females tested, three swam to the cage containing sterilized,
spermiating males, two swam to the cage containing sterilized, prespermiating males, and
ﬁve did not choose a stimulus source. The distributions of choices differed signiﬁcantly
between ovulating and preovulating females (P = 0.024). Although the behavior was not
quantiﬁed, we observed ovulating females swimming repetitively around and against the
cage containing sterilized, spermiating males for l h. The three preovulating females that
swam to the sterilized, spermiating male cage and the two that swam to the sterilized,
prespermiating male cage did not display this behavior, but paused brieﬂy at the cages
and continued swimming upstream.

In EOG experiments the chemical stimuli released by sterilized (N = 8) and
nonsterilized (N = 8), spermiating males induced similar responses from the olfactory
epithelium of adult females, but chemical stimuli from nonsterilized, prespermiating
males (N = 4) did not at equivalent concentrations (Figure 1). Both sterilized and

nonsterilized, spermiating male conditioned waters had detection thresholds of

112

Figure 1. (a) Representative electro-olfactogram (EOG) responses of female sea
lampreys (Petromyzon marinus) to chemical stimuli from sterilized, spermiating males
(SSM, diamonds), non-sterilized, spermiating males (SM, open circles), and non-
sterilized, pre-sperrniating males (PSM, ﬁlled circles). Std designates the response to a
10'5 M L-arginine standard and Con the response to a blank water control. Numbers
along the x-axis indicate the logarithmic value of the dilution from the original
conditioned water collected by holding one male in 10 l for 4 h. (b) Female sea lamprey
EOG dose-response relationships to SSM (N = 8), SM (N = 8), and PSM (N = 4)
chemical stimuli. Responses are measured as a percentage of the response to a 10'5 M L-
arginine standard. Vertical bars represent one standard error.

113

 

 

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114

approximately 105 times dilution. The detection threshold for nonsterilized,
prespermiating male chemical stimuli was approximately 102 times dilution, one
thousand times more concentrated than the dilution required for detection of sterilized
and nonsterilized, spermiating male chemical stimuli.

Cross—adaptation experiments demonstrated that female olfactory responses were
affected by continuous exposure to spermiating male chemical stimuli (ANOVA; P <
0.01). Although the responses to L-arginine standard did not signiﬁcantly change before
and during adaptation (Student’s t test; P > 0.05), there were substantial differences
among responses to sterilized and nonsterilized, spermiating male chemical stimuli
before and during adaptation (Student’s t tests; P < 0.01). Both sterilized and non-
sterilized, spermiating male chemical stimuli suppressed olfactory responsiveness to
themselves and each other when used as adapting stimuli (Figure 2a). For example, when
sterilized spermiating male chemical stimuli (Figure 2b; N = 5) were used as the adapting
stimuli, the initial responses were 2.17 3: 0.23 mV for sterilized, spermiating male
chemical stimuli and 1.52 i 0.39 mV for nonsterilized, spermiating male chemical
stimuli, while during adaptation, the responses of sterilized, spermiating male chemical
stimuli to itself was 0.08 i 0.02 mV and to nonsterilized, spermiating male chemical
stimuli was 0.18 i 0.13 mV. When nonsterilized, spermiating male chemical stimuli
were used as the adapting stimulus (Figure 2c; N = 5), the same pattern of olfactory
suppression occurred for each chemical stimulus.

Negative and positive fast atom bombardment mass spectrometry showed that the
base peak (the most abundant molecule) of crude extracts of all three samples of water

conditioned with sterilized, spermiating males had a molecular weight of 472 Dalton, the

115

Figure 2. (a) Representative electro-olfactogram (EOG) responses of female sea
lampreys (Petromyzon marinus) to chemical stimuli from sterilized spermiating male
(SSM) and non-sterilized spermiating male (SM) before (white bars) and during (shaded
bars) adaptation to a SSM chemical stimuli. Std designates the response to a 10'5 M L-
arginine standard and Con the response to a blank water control. Female EOG responses
to a 10'5 M L-arginine standard, SSM and SM chemical stimuli both before and during
adaptation to SSM (b) and SSM (c) chemical stimuli were measured. Vertical bars
represent one standard deviation.

116

 

 

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117

same as extracts from the three samples of water conditioned with nonsterilized,
spermiating males (Figure 3a, 3b). The isotopic patterns of sterilized and nonsterilized,
spermiating males are virtually identical and no other major peaks are present in either

spectrum.

118

(a)
100 -

80 4
472
60 -

40-

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400 420 440 460 480 500

100 -

Relative abundance (%)

80 r
472
60 -
40 -

20-

 

 

 

Figure 3. Negative and positive fast atom bombardment mass spectrometry (10 KV)
spectrum of (a) extracts of water conditioned with non-sterilized spermiating male sea
lampreys (Petromyzon marinas) and (b) extracts of water conditioned with sterilized
spermiating males.

119

DISCUSSION

The combined results of all four experiments show that bisazir-sterilized male sea
lampreys during spermiation are capable of attracting ovulating females, and this
attraction is mediated through the release of a sex pheromone in an amount similar to that
released by nonsterilized males during spermiation. In the ﬁrst experiment, when placed
in a two-choice maze, ovulating females both preferred and spent more time searching in
the treatment side of the maze containing chemical stimuli from sterilized, spermiating
males. These results are nearly identical to those of Li et al. (2002) where 22 of 22 and
eight of eight ovulating females preferred and spent more time searching in the side of
the maze conditioned with nonsterilized, spermiating males and 12 of 15 and 7 of 7
ovulating females preferred and spent more time searching in the side conditioned with
nonsterilized, spermiating male washings. Searching behavior could be the mechanism
underlying the attraction response that leads female sea lampreys to male partners in a
spawning stream. Sea lampreys oﬁen nest in rapids and chemical stimuli from nesting
animals are ﬂushed downstream (Applegate 1950). Individuals responding to chemical
stimuli by searching and swimming against the current stand a better chance of reaching
the nesting individuals upstream (Li et al. 2002). Further, ovulating, but not preovulating
females were attracted to sterilized, spermiating males in their natural spawning habitat,
further demonstrating that only ovulating females that are ready to spawn are attracted to
spermiating males. It is notable that in a previous study conducted in the same stream

section under similar conditions, 9 of 13 ovulating females chose the side conditioned

120

with ﬁve nonsterilized, spermiating males (Li et al. 2002), whereas in this study 10 of 15
ovulating females chose the side conditioned with ﬁve sterilized, spermiating males.

The virtually identical levels of behavioral responses observed in the ﬁrst
experiment may be attributable to the virtually identical levels of sex pheromone
production and release between sterilized and nonsterilized, spermiating males, as
demonstrated in experiments 2 and 3. In the second experiment, EOG results showed
that sterilized and nonsterilized, spermiating male chemical stimuli produced similar
dose-response relationships in females, demonstrating that the chemical stimuli from both
types of animals have the same potency. The olfactory potency of nonsterilized,
spermiating male chemical stimuli was ﬁrst observed by Bjerselius et al. (1995). Our
results conﬁrm the potency and show that sterilized males can be just as potent. Also,
EOG cross-adaptation experiments showed that chemical stimuli from sterilized and
nonsterilized, spermiating males suppressed olfactory responsiveness to each other and to
themselves after olfactory adaptation to one had occurred, demonstrating that water
conditioned with sterilized and nonsterilized, spermiating males are of the same quality in
terms of their olfactory stimulatory effectiveness.

Our fast atom bombardment mass spectrometry analysis further showed that the
same pheromonal molecule is responsible for the behavioral and electrophysiological
responses elicited by chemical stimuli from both sterilized and nonsterilized, spermiating
males. The most abundant molecule contained in extracts of water conditioned with
sterilized and nonsterilized, spermiating males has the same molecular weight and
isotopic patterns that are characteristic of 7a, 1211, 24-trihydroxy 5a-cholan-3-one 24-

sulfate, the molecule demonstrated to induce behaviors identical to those induced by

121

nonsterilized, spermiating males (Li et al. 2002) and by sterilized, spermiating males. All
four of our experiments demonstrate the biosynthesis and release of a sex pheromone by
sterilized, spermiating males. The close similarity of our results to that of Li et al. (2002)
also demonstrate that the mechanisms regulating pheromone production and release were
intact in males sterilized by bisazir treatment.

For a sterile male release technique to be effective, it is essential that the
sterilization process does not negatively affect the mating competency of sterilized
individuals (Knipling 1964). Previous ﬁeld tests demonstrated that sterile male sea
lampreys displayed nest-building behaviors and obtained mates (Hanson and Manion
1978). Our study expanded on these ﬁndings and further demonstrated that the male sex
pheromone signaling system is not affected by bisazir treatment. Bisazir may induce
lethal mutations in gametes (Hanson and Manion 1980) that cause premature death in
heterozygotes if dominant, and in homozygotes if recessive. Spawning behaviors of sea
lampreys, on the other hand, are under direct regulation by the central nervous system,
which in turn is inﬂuenced by the hypothalamus-pituitary gland-gonad axis (Sower
1990). Since the release of sex pheromone coincides with spermiation (Li et al. 2002), a
process also regulated by the hypothalamus-pituitary gland-gonad axis (Sower 1990), it is
possible that sex pheromone release is also regulated by this axis. It is not likely that the
dosage of bizasir injected for sterilization will affect this system, and thus, reproductive
behavior, pheromone release, and ultimately mating efﬁcacy. Further, it appears that the
slightly higher than expected levels of egg viability from the sterile male release
technique (Bergstedt et al. 2003) is not due to the effect of bisazir treatment on

pheromone synthesis and release.

122

Recently, the potential upregulation of pheromone biosynthesis and release was
proposed for the sterile male release technique program of sea lamprey management (Li
et a1. 2003). The concept is that sterilized males would be induced to synthesize and
release the sex pheromone at higher concentrations, for longer periods of time, or both.
A similar concept, where cues (similar to natural pheromonal compounds) are
synthesized to be more potent, have been developed for the oriental fruit ﬂy (Bactrocera
dorsalis) and applied in their control (Lanier 1990). If a technique to upregulate the sex
pheromone in sea lampreys can be developed, it would potentially improve the efﬁcacy
of the current sterile male release technique, which is mainly limited by the number of
males that can be collected for sterilization and release (Twohey et al. in 2003b).

Numerous invasive ﬁsh species are currently affecting ﬁsh communities in the
Great Lakes (Michigan Sea Grant 1994), large lake ecosystems worldwide (Hall and
Mills 2000), and other aquatic ecosystems across North America (Courtenay et al. 1986).
Resistance to widespread use of nonselective pesticides severely limits efforts to control
these or any invasive ﬁsh species. Also, alternatively, nonchemical control methods have
been developed for other pests, but most are not easily transferred to ﬁsh. Sterile male
release may provide an alternative means to control invasive ﬁsh species if it can be
demonstrated that bisazir treatment does not affect reproductive function, including

production and release of sex pheromones in these ﬁsh.

123

ACKNOWLEDGEMENTS

We thank the staffs of the US. Geological Survey Hammond Bay Biological
Station and the US. Fish and Wildlife Service Marquette Biological Station for their
assistance with this project. We also thank Dolly Trump and Lydia Lorenz for the use of
their private land as a ﬁeld study site. The Department of Fisheries and Oceans, Ontario,
Canada supplied radiotelemetry equipment and Dr. Douglas Gage of Michigan State
University provided the facility for and expertise in chemical analysis. This research was
supported by the Great Lakes Fishery Commission. This article is Contribution 1224 of

the US. Geological Survey, Great Lakes Science Center.

124

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Michigan. US. Fish. Wildl. Serv. Spec. Sci. Rep. Fish. Serv. no. 55

Bergstedt, R.A., McDonald, R.B., Twohey, M.B., Mullett, K.M., Young, R.J., and
Heinrich, J .W. 2003. Reduction in reproductive success of sea lampreys through
release of sterilized males. J. Great Lakes Res. In press.

Bjerselius, R., Li, W., Sorensen, P.W., and Scott, AP. 1996. Spermiated male sea
lamprey release a potent sex pheromone. In Proceedings of the ﬁfth international
symposium on the reproductive physiology of ﬁsh. Edited by P. Thomas and F. Goetz.
Austin, TX, pp. 271.

Coble, D.W., Bruesewitz, R.E., Fratt, T.W., and Schreirer, J.W. 1990. Lake trout, sea
lamprey, and overﬁshing in the upper Great Lakes: a review and reanalysis. Trans.
Am. Fish. Soc. 119: 985-995.

Courtenay, W.R., Hensley, D.A., Taylor, J .N., and McCann, J .A. 1986. Distribution of
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Fishes. Edited by CH. Hocutt and EC. Wiley. New York, Wiley. pp. 675-698.

Getchell, TV. 1974. Electrogenic sources of slow voltage transients recorded from frog
olfactory epithelium. J. Neurophysiol. 37: 1115-1130.

Hall, SR. and Mills, EL. 2000. Exotic species in large lakes of the world. Aquat.
Ecosyst. Health Manage. 3(1): 105-135.

Hanson, L.H. and Manion, PJ. 1980. Sterility method of pest control and its potential

role in an integrated sea lamprey (Petromyzon marinus) control program. Can. J. Fish.
Aquat. Sci. 37: 2108-2117.

Hanson, L.H. and Manion, PJ. 1978. Chemosterilization of the sea lamprey (Petromyzon
marinas). Great Lakes Fish. Comm. Tech. Rep. No. 29.

Kelso, J .R.M. and Gardner, W.M. 2000. Emmigration, upstream movement, and habitat
use by sterile and fertile sea lampreys in three Lake Superior tributaries. N. Am. J.
Fish. Manage. 20: 144-153.

Kitchell, J .F. 1990. The scope for mortality caused by sea lamprey. Trans. Am. Fish. Soc.
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Knipling, ER 1964. The potential role of the sterility method for insect population
control with special reference to combining this method with conventional methods.
US. Dept. Agric. Res. Serv. ARS-33-98.

Lanier, G. N. 1990. Principles of attraction-annihilation: mass trapping and other means.
In Behavior-modifying chemicals for pest management: applications of pheromones
and other attractants. Edited by RL. Ridgway, R.M. Silverstein and M. N. Inscoe.
New York: Marcel Kekker, pp 25-46.

Li, W. 1994. The olfactory biology of adult sea lamprey (Petromyzon marinus). Ph. D
thesis. University of Minnesota.

Li, W. and Sorensen, P.W. 1997. Four independent olfactory receptor sites for bile acids,
putative migratory pheromones, in adult sea lamprey (Petromyzon marinus). J. Comp.
Physiol. A. 180(4): 429-438.

Li, W., Sorensen, P.W., and Gallaher, DD. 1995. The olfactory system of migratory
adult sea lamprey (Petromyzon marinus) is speciﬁcally and acutely sensitive to
unique bile acids released by conspeciﬁc larvae. J. Gen Physiol. 105: 567-587.

Li, W., Scott, A.P., Sieﬂtes, M.J., Yan, H., Liu, Q, Yun, S-S, and Gage, DA. 2002. Bile
Acid Secreted by Male Sea Lamprey that Acts as a Sex Pheromone. Science 296: 138-
141.

Li, W., Siefkes, M.J., Scott, AP, and Teeter, J .H. 2003. Sex pheromone
communication in the sea lamprey: implications for integrated management. J. Great
Lakes Res. In press.

Liley, NR. 1982. Chemical communication in ﬁsh. Can. J. Fish. Aquat. Sci. 39: 22-35.

Michigan Sea Grant, University of Michigan, Ann Arbor, USA Sea Grant Program. 1994.
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Ottoson, D. 1956. Analysis of the electrical activity of the olfactory epithelium. Acta
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Rao, P.V. 1998. Statistical Research Models in the Life Sciences. Brooks /Cole
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Schleen, L.P., Christie, G.C., Heinrich, J.W., Bergstedt, R.A., Young, R.J., Morse, T.J.,
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in the St. Mary’s River: a case study in integrated and coordinated ﬁsheries
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Smith, BR. 1971. Sea lamprey in the Great Lakes of North America. In The biology of
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pp. 207-247.

Smith, BR. and Tibbles, J .J . 1980. Sea lamprey (Petromyzon marinus) in Lake Huron,
Michigan, and Superior: history of invasion and control, 1936-1978. Can. J. Fish.
Aquat. Sci. 37: 1780-1801.

Solomon, DJ. 1977. A review of chemical communication in freshwater ﬁsh. J. Fish.
Biol. 11: 363-376.

Sower, SA. 1990. Neuroendocrine control of reproduction in lampreys. Fish Physiol.
Biochem. 8(5): 365-374.

Spangler, G.R., Robson, D.S., and Regeir, HA. 1980. Estimates of lamprey-induced
mortality in Whiteﬁsh, Coregonus clupeaformis. Can. J. Fish. Aquat. Sci. 37(11) 2146-
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Stacey, N.E., Cardwell, J.R., Liley, N.R., Scott, AR, and Sorensen, P.W. 1994.
Hormones as sex pheromones in ﬁsh. In Perspectives in ComparativeEndocrinology.
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Twohey, M.B., Hanson, L.H., Heinrich, J .W., Seelye, J .G., Bergstedt, R.A., McDonald,
RB, and Christie, G.C. 2003a. History and development of the sterile male release
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Twohey, M.B., Sorensen, P.W., and Li, W. 2003b. How might new knowledge of
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127

SUMMARY OF DISSERTATION

At the onset of this research the function of the male sea lamprey sex pheromone
had been partially characterized, and its speciﬁcity and identity had been determined.
The male sex pheromone was shown to be released by spermiating males and to attract
ovulating females (Pre-spermiating males were not attractive to ovulating females and
pre-ovulating females were not attracted to spermiating males). The spermiating male
sex pheromone was found to be the bile acid 701, 12a, 24-trihydroxy-5cr-cholan-3-one-24
sulfate (3-keto petromyzonol sulfate or 3kPZS). Overall, this was the ﬁrst male sex
pheromone identiﬁed in ﬁsh and appeared to be capable of functioning over relatively
long distances.

This research examined several aspects of sea lamprey sex pheromone
communication in order to elucidate the intricate mechanisms whereby mature males
attract mature females. In the laboratory, I determined the site of synthesis and excretion
of 3kPZS and the potency and speciﬁcity of 3kPZS to female olfactory organs in order to
understand the physiological processes for 3kPZS to realize its signaling potential. In the
interim, the effectiveness of 3kPZS and water conditioned with spermiating males was
demonstrated in a natural spawning environment.

In chapter 1, techniques including behavioral assays, electro-olfactograms,
biochemical analyses, and immunocytochemistry were used to show that 3kPZS was
produced in the liver and released exclusively through the gills of spermiating males. For
a sex pheromone signaling system to function over long distances a means of achieving

biologically relevant concentrations downstream needs to be developed. The gills have

128

 

an enormous surface area and offer a potentially efﬁcient site for 3kPZS exchange with
the environment. Therefore, it appears that sea lampreys have evolved a specialized sex
pheromone release mechanism that is capable of releasing large amounts of 3kPZS.

In chapter 2, electro-olfactograms were used to show that 3kPZS is a potent
odorant that is detected at a concentration of 10'12 M and that 3kPZS is discriminated
from other conspeciﬁc bile acids by the olfactory organs of females. These results also
lend evidence to support that the male sex pheromone functions over long distances. Sea
lampreys spawn in fast-ﬂowing water and 3kPZS that is released by males will be
quickly diluted. Also, sea lampreys spawn in streams that contain larvae (as well as other
adults) that release bile acids similar in structure to 3kPZS. The ability to detect 3kPZS
at extremely low concentrations and to discriminate it from similar odorants is necessary
in order for 3kPZS to ﬁrnction as a sex pheromone over long distances.

In chapter 3, in-stream behavioral assays using radio telemetry showed that both
10"2 M 3kPZS and water conditioned with spermiating males (in which the 3kPZS
concentration was estimated to be 2 x 10"2 M) were able to attract ovulating females 65
m upstream to the point of odorant introduction. These results are direct evidence that
the male sex pheromone is able to function over long distances. Also, the effectiveness
of 10‘l2 M 3kPZS in these behavioral experiments complement the detection thresholds
found in chapter 2.

Not only do these results conﬁrm the hypothesis that 3kPZS is a male sex
pheromone that can function over long distances and further elucidate the mechanisms
whereby mature males attract mature females, they also have the potential to impact sea

lamprey management in the Great Lakes. By knowing the production and release site of

129

3kPZS and how females detect conspeciﬁc bile acids, it may be possible to use this
knowledge to disrupt the signaling system either by preventing males from releasing
3kPZS or preventing females from detecting 3kPZS. Furthermore, by knowing that the
sex pheromone functions in a natural environment it appears feasible to develop trapping
techniques to remove females from the spawning grounds. Also, further exploring the
production mechanisms of 3kPZS may yield a productive and cheap way to synthesize
3kPZS, which could be used for potential management practices like trapping.

The male sex pheromone may also be used to augment current sea lamprey
control strategies such as the sterile male release technique. If sex pheromone production
in sterilized males can be upregulated, sterilized males may be more competitive for
mates. However, whether or not the sterilization process affects sex pheromone release
was unknown. Chapter 4 of this dissertation describes a multi-disciplinary approach
including behavioral assays, electro-olfactograms and mass spectrometry that
demonstrated that the sterilization process does not affect sex pheromone release.

In addition to the possibility of developing new concepts and techniques for
integrated sea lamprey management, these results advance our understanding of sex
pheromone communication in ﬁsh and vertebrates. This is the ﬁrst male sex pheromone
and ﬁrst bile acid sex pheromone identiﬁed in ﬁsh and has been shown to function at low
concentrations in a natural environment over relatively long distances. Moreover this
study offers the ﬁrst example that gills contain the machinery to and do release a sulfated
sex pheromone. In the long run, information on how this sex pheromone is produced and
released and how adult sea lampreys smell bile acids produced by conspeciﬁcs will bring

beneﬁts outside of sea lamprey management that are not immediately apparent.

130

APPENDIX

PERMISSION TO USE PUBLISHED MATERIAL

131

Permission to use the following article

Sieﬂces, M.J., Scott, A.P., Zielinski, B., Yun, S.-S., and Li, W. 2003. Male sea lampreys,

Petromyzon marinus L., excrete a sex pheromone from gill epithelia. Biology of
Reproduction, 69:125-132.

September 3, 2003

Ms. Judith Jansen

Society for the Study of Reproduction
1619 Monroe Street

Madison, WI 53711-2063

Dear Ms. Jansen:

I recently spoke with a member of your staff about gaining your permission to use a recently
published article in my Ph.D. dissertation. The citation is:

Siefkes, MJ, Scott, AP, Zielinski, B, Yun, S-S, and Li, W. 2003. Male sea
lampreys, Petromyzon marinas L., excrete a sex pheromone from gill epithelia.
Biol. Reprod. 69:125-132.

My dissertation will be published for the Michigan State University Library in December of
2003. The above-mentioned paper will be appropriately cited within this dissertation. Biology of
Reproduction will maintain the copyright to the above paper.

Ifyou need any other statements from me to make this ofﬁcial, please let me know. Please send
the letter of permission to:

Michael J. Siefkes

Department of Fisheries and Wildlife
Michigan State University

13 Natural. Resources Building

East Lansing, MI 48824

Thanks for your time.

Michael Siefkes

 

Pennission granted bythe Societytorthe Studyof
Reproduction, lnc.. provided that the original
publication is appropriately cited.

 

 

 

 

132

Permission to use the following article

Siefkes, M.J. and Li, W. In press. 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.

Dear Alice,

I recently had a manuscript accepted for publication in one of your journals (Journal of
Comparative Physiology A). I am ﬁnishing up my PhD and need to use this paper in my
thesis. How do I obtain permission to use this accepted paper? Your quick response will
be greatly appreciated. Thanks.

Michael J. Sieﬂces

Department of Fisheries and Wildlife
Michigan State University

13 Natural Resources

East Lansing, MI 48824

Phone: (517)353-7981

Fax: (517)432-1699

Dear Michael,

Thank you for your e-mail of November 25, 2003. We are pleased to grant you the
permission requested, provided full credit (Springer journal title, article title, name(s) of
author(s), volume, page numbers, year of publication, Springer copyright notice) is given
to the publication in which the material was originally published.

With kind regards,

Rosita

******************

Rosita Sturm (Ms.)

Rights & Permissions
Springer-Verlag GmbH & Co. KG
Tiergartenstrasse 17

69121 Heidelberg

GERMANY

Tel: 4+ 49 (0) 6221 - 487 8228
Fax: ++ 49 (0) 6221 - 487 8100
e-mail: Sturm@springer.de
internet: www.springer.de/rights

133

Permission to use the following article

Sieﬂces, M. J ., Bergstedt, R. A., Twohey, M. B., and Li, W. 2003. Chemosterilization of
male sea lampreys (Petromyzon marinus) does not affect sex pheromone release.
Canadian Journal of Fisheries and Aquatic Sciences, 60:23-31.

Paul,

The editorial ofﬁce of CJFAS told me to contact you about getting permission to use the
following published manuscript in my PhD dissertation:

Sieﬂtes MJ, Bergstedt RA, Twohey MB, and Li W. 2003. Chemosterilization of male sea
lampreys does not affect sex pheromone release. Can. J. Fish. Aquat. Sci. 60: 23-31.

If you could let me know the procedures for including this in my dissertation, it would be
greatly appreciated. Thanks for your time.

Michael J. Sieﬂtes

Department of Fisheries and Wildlife
Michigan State University

13 Natural Resources

East Lansing, MI 48824

Phone: (517)432-1141

Fax: (517)432-1699

Dear Michael

Permission is granted for use of the material, as descibed below, provided that
acknowledgement is given to the source.

Sincerely,

Paul McClymont

Business Manager

NRC Research Press

Tel: 613-993-9093

Fax: 613-952-7656

E-Mail: paul.mcclymont@nrc.ca

----- Original Message-----

From: Michael Sieﬂces [mailtozsieﬂtesm@msu.edu]

Sent: September 2, 2003 2:51 PM

To: paul.mcclymont@nrc.ca

Subject: Permission to include a published manuscript in my PhD dissertation

134