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02079 9361

This is to certify that the

thesis entitled

SPERMIATING MALE SEA LAMPREYS
RELEASE A SEX PHEROMONE THAT ATTRACTS
POST-OVULATORY FEMALE SEA LAMPREYS

presented by

Michael J. Siefkes
has been accepted towards fulfillment

of the requirements for

M.S. degreg in Fisheries & WIldllfe

1'2.

Major p 850:

 

 

 

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Michigan State
University

 

 

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TO AVOID FINES return
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0X to remove this checkout from your record.
on or before date due.
her due date if requested.

 

 

DATE DUE ‘

DATE DUE

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SPERMIATING MALE SEA LAMPREYS RELEASE A SEX PHEROMONE THAT
ATTRACTS POST-OVULATORY FEMALE SEA LAMPREYS

BY

Michael J. Siefkes

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

Department of Fisheries and Wildlife

2000

ABSTRACT

SPERMIATING MALE SEA LAMPREYS RELEASE A SEX PHEROMONE THAT
ATTRACTS POST-OVULATORY FEMALE SEA LAMPREYS

BY

Michael J. Siefkes

Behavioral observations suggested that a jawless fish, the sea
lamprey, Petromyzon marinas releases sex pheromones that
influence the behavior of conspecifics. Further,
electrophysiological studies showed that male sea lampreys
release a potent odorant during spermiation. I hypothesized that
this odorant is a sex pheromone and examined its effects on the
behavior of adult sea lampreys. My results show that this
odorant specifically influences the behavior of post—ovulatory
females. When placed in a two-choice maze, post-ovulatory
females spent more time and showed increased searching or
swimming activity in the side containing a spermiating male
odorant. Similar behavioral responses were not observed in male
and pre-ovulatory female sea lampreys. Also, in a natural
spawning stream, post—ovulatory females located and swam to cages
containing spermiating males, but pre—ovulatory females did not.
I conclude that this odorant is a sex pheromone that specifically
attracts post-ovulatory female sea lampreys through increased
searching behavior and further speculate that it ultimately

functions to coordinate spawning.

I would like to dedicate this thesis to my family, friends, and
Rebecca Otto. Their continuous and enthusiastic support is

essential in my quest to further my education.

— iii -

ACKNOWLEDGEMENTS

First I would like to thank my advisor Dr. Weiming Li and the
members of my graduate committee, Mr. Roger Bergstedt, Dr. Doug
Gage, and Dr. Dan Hayes for their guidance and support. Second,
I am grateful to the staff of the United States Geological
Survey, Hammond Bay Biological Station for their advice and use
of space, materials and equipment and Mr. Mike Twohey and the
staff at the United States Fish and Wildlife Service, Marquette
Biological Station for the sea lampreys used in experiments.
Third, I would like to thank Dr. John Kelso of the Department of
Fisheries and Oceans, Ontario, Canada for his equipment and
advice on radio telemetry. Next, I would like to thank Mrs.
Dolly Trump and Lydia Lorenz for the use of their land adjacent
to the Ocqueoc River and all the summer interns that helped me to
complete this project. Lastly, I would like to express thanks to
the Great Lakes Fishery Commission for providing funding and

enthusiastic support for this work.

-iv-

TABLE OF CONTENTS

LIST OF TABLES.
LIST OF FIGURES.
INTRODUCTION.

METHODS
Collection and Maintenance of Animals.
Behavioral Preference Tests.
Searching Behavior Tests.
Behavior in a Spawning Stream.

RESULTS
Behavioral Preference Tests.
Searching Behavior Tests.
Behavior in a Spawning Stream.

DISCUSSION.

APPENDICES
Appendix A. Indoor Preference Test Raw Data.
Appendix B. Outdoor Preference Test Raw Data.
Appendix C. Indoor Searching Behavior Raw Data.
Appendix D. Outdoor Searching Behavior Raw Data.
Appendix E. Spawning Stream Behavior Raw Data.

REFERENCES.

.vi

.xvi

.10

11

15

.18

22

.25

30

.51
.58

65
72

.75

Table 1.

Table 2.

Table 3.

LIST OF TABLES

Number of adult sea lampreys of specific sex and
maturity choosing the treatment (odorant) or control
(no odorant) side of an indoor two-choice maze. The
treatment side used spermiating male (SM), non-
spermiating male (NSM), post-ovulatory female (OF),
and pre-ovulatory female (NOF) sea lampreys and also
water collected from spermiating males (SMW) as odor
sources. Test subjects were spermiating males (SM),
non—spermiating males (NSM), post-ovulatory females
(OF), and pre-ovulatory females (NOF). Statistical
significance was determined with a Wilcoxon Signed
Ranks Test (2-tailed) using indices of preference (see
Appendix A).

Number of adult sea lampreys of specific sex and
maturity choosing the treatment (odorant) or control
(no odorant) side of an outdoor two-choice maze. The
treatment side used spermiating male (SM) and non-
spermiating male (NSM) sea lampreys and also water
collected from spermiating males (SMW) as odor
sources. Test subjects were spermiating males (SM),
non-spermiating males (NSM), post-ovulatory females
(OF), and pre-ovulatory females (NOF). Statistical
significance was determined with a Wilcoxon Signed
Ranks Test (2-tailed) using indices of preference (see
Appendix B).

Number of adult sea lampreys of specific sex and
maturity spending more time searching in the treatment
(odorant) or control (no odorant) side of an indoor
two-choice maze. The treatment side used spermiating
male (SM) and non-spermiating male (NSM) sea lampreys
and also water collected from spermiating males (SMW)
as odor sources. Test subjects were spermiating males
(SM), non-spermiating males (NSM), post-ovulatory
females (OF), and pre—ovulatory females (NOF).
Statistical significance was determined with a
Wilcoxon Signed Ranks Test (2-tailed) using indices of
searching (see Appendix C).

-Vi-

Table 4.

Table 5.

Table 6.

Table 7.

LIST OF TABLES (cont’d)

Number of adult sea lampreys of specific sex and
maturity spending more time searching in the treatment
(odorant) or control (no odorant) side of an outdoor
two—choice maze. The treatment side used spermiating
male (SM) and non—spermiating male (NSM) sea lampreys
and also water collected from spermiating males (SMW)
as odor sources. Test subjects were spermiating males
(SM), non-spermiating males (NSM), post-ovulatory
females (OF), and pre-ovulatory females (NOF).
Statistical significance was determined with a
Wilcoxon Signed Ranks Test (2-tailed) using indices of
searching (see Appendix D).

Number of post-ovulatory (OF) and pre-ovulatory (NOF)
female sea lampreys swimming to spermiating males
(SM), non-spermiating males (NSM), or staying at an
intermediate (Int.) position within a section of a
known sea lamprey spawning stream. The distribution of
choices were significantly different between post—
ovulatory and pre-ovulatory females (Fisher’s Exact
Test, 2—Tail, P = 0.024).

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for post—ovulatory female test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for pre-ovulatory female test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

-vfi-

Table 8.

Table 9.

Table 10.

Table 11.

Table 12.

LIST OF TABLES (Cont’d)

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for spermiating male test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for non—spermiating male test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for post-ovulatory female test subjects
exposed to a spermiating male washing odorant.
Positive I values indicate an attraction and negative
I values indicate a repulsion. The absolute values of
the I values were analyzed with a Wilcoxon Signed
Ranks Test (two-tailed) to determine overall
preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for post—ovulatory female test subjects
exposed to a non—spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for pre-ovulatory female test subjects
exposed to a non-spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

- viii -

Table 13.

Table 14.

Table 15.

Table 16.

Table 17 .

LIST OF TABLES (cont’d)

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for spermiating male test subjects
exposed to a non-spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed using a Wilcoxon Signed Ranks
Test (two-tailed) to determine overall preference
behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for non—spermiating male test subjects
exposed to a non-spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for post-ovulatory female test subjects
exposed to a post-ovulatory female odorant. Positive
I values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of

preference (I) for pre-ovulatory female test subjects
exposed to a postrovulatory female odorant. Positive
I values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I

values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for spermiating male test subjects
exposed to a post-ovulatory female odorant. Positive
I values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

-ix-

Table 18.

Table 19.

Table 20.

Table 21.

Table 22.

LIST OF TABLES (cont’d)

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of

preference (I) for non-spermiating male test subjects
exposed to a post-ovulatory female odorant. Positive
I values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I

values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for post-ovulatory female test subjects
exposed to a pre-ovulatory female odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for pre—ovulatory female test subjects
exposed to a pre—ovulatory female odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for spermiating male test subjects
exposed to a pre-ovulatory female odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of
preference (I) for non-spermiating male test subjects
exposed to a pre-ovulatory female odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Table 23.

Table 24.

Table 25.

Table 26.

Table 27.

LIST OF TABLES (cont’d)

Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of
preference (I) for post-ovulatory female test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of
preference (I) for pre-ovulatory female test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of
preference (I) for spermiating male test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of
preference (I) for non—spermiating male test subjects
exposed to a spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of
preference (I) for post—ovulatory female test subjects
exposed to a spermiating male washing odorant.
Positive I values indicate an attraction and negative
I values indicate a repulsion. The absolute values of
the I values were analyzed with a Wilcoxon Signed
Ranks Test (two-tailed) to determine overall
preference behavior.

-xi-

Table 28.

Table 29.

Table 30.

Table 31.

LIST OF TABLES (cont’d)

Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of
preference (I) for post—ovulatory female test subjects
exposed to a non-spermiating male odorant. Positive I
values indicate an attraction and negative I values
indicate a repulsion. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test
(two—tailed) to determine overall preference behavior.

Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and
indices of searching (I) for post-ovulatory female
test subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-
tailed) to determine overall searching behavior.

Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and
indices of searching (I) for pre—ovulatory female test
subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-
tailed) to determine overall searching behavior.

Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and
indices of searching (I) for spermiating male test
subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-
tailed) to determine overall searching behavior.

-xfi-

Table 32.

Table 33.

Table 34.

Table 35.

LIST OF TABLES (cont’d)

Time spent (seconds) searching in each Side of the
indoor mazes before and after odorant introduction and
indices of searching (I) for non-spermiating male test
subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-
tailed) to determine overall searching behavior.

Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and
indices of searching (I) for post-ovulatory female
test subjects exposed to a spermiating male washing
odorant. Positive I values indicate more searching in
treatment side and negative I values indicate more
searching in control side. The absolute values of the
I values were analyzed with a Wilcoxon Signed Ranks
Test (two—tailed) to determine overall searching
behavior.

Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and
indices of searching (I) for post-ovulatory female
test subjects exposed to a non-spermiating male
odorant. Positive I values indicate more searching in
treatment side and negative I values indicate more
searching in control side. The absolute values of the
I values were analyzed with a Wilcoxon Signed Ranks
Test (two-tailed) to determine overall searching
behavior.

Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction
and indices of searching (I) for post-ovulatory female
test subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two—
tailed) to determine overall searching behavior.

- xiii -

Table 36.

Table 37.

Table 38.

Table 39.

LIST OF TABLES (Cont’d)

Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction
and indices of searching (I) for pre-ovulatory female
test subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two—
tailed) to determine overall searching behavior.

Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction
and indices of searching (I) for spermiating male test
subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two—
tailed) to determine overall searching behavior.

Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction
and indices of searching (I) for non-spermiating male
test subjects exposed to a spermiating male odorant.
Positive I values indicate more searching in treatment
side and negative I values indicate more searching in
control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-
tailed) to determine overall searching behavior.

Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction
and indices of searching (I) for post-ovulatory female
test subjects exposed to a spermiating male washing
odorant. Positive I values indicate more searching in
treatment side and negative I values indicate more
searching in control side. The absolute values of the
I values were analyzed with a Wilcoxon Signed Ranks
Test (two—tailed) to determine overall searching
behavior.

-xiv-

Table 40.

Table 41.

Table 42.

LIST OF TABLES (cont’d)

Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction
and indices of searching (I) for post-ovulatory female
test subjects exposed to a non-spermiating male
odorant. Positive I values indicate more searching in
treatment side and negative I values indicate more
searching in control side. The absolute values of the
I values were analyzed with a Wilcoxon Signed Ranks
Test (two—tailed) to determine overall searching
behavior.

Post—ovulatory females choosing spermiating males
(SM), non-spermiating males (NSM) or staying at an
intermediate position (Int.) within a spawning stream
study section. Times in which females reached either
spermiating or non-spermiating males are recorded in
minutes. Time is not applicable (NA) to those females
choosing an intermediate position.

Pre-ovulatory females choosing spermiating males (SM),
non-spermiating males (NSM) or staying at an
intermediate position (Int.) within a Spawning stream
study section. Times in which females reached either
spermiating or non-spermiating males are recorded in
minutes. Time is not applicable (NA) to those females
choosing an intermediate position.

-xv-

Figure 1.

Figure 2.

Figure 3.

LIST OF FIGURES

Design of indoor and outdoor two-choice mazes used in
preference and searching behavior tests. Arrows
indicate water flow. Dotted lines indicate flow
through plastic mesh. Dashed line indicates low—head
laminar flow device.

Two-choice maze used in outdoor experiments. Water
was pumped into the head of the maze and allowed to
flow through back into the river. A single sea
lamprey was introduced and its behavior in the maze
was monitored both before and after an odorant from
sea lampreys of a specific sex and maturity was
introduced.

The upstream portion of the study section including an
island that divides the river and the odor cages used
in spawning stream behavior experiments. Post-
ovulatory and pre-ovulated female sea lampreys were
fitted with radio tags and their behavior monitored in
response to spermiating and non—spermiating male
odorants.

-xvi-

INTRODUCTION

In many species of fish, pheromones produced during final
maturation have been shown to function as both behavioral and
physiological agents in the synchronization of mating (Partridge
et a1. 1976; Liley 1982; Stacey & Sorensen 1991; Stacey et al.
1994; Scott & Vermeirssen 1994). Specifically, sex pheromones
have been shown to stimulate vitellogenesis (Van Weerd and Kamen
1998) and ovulation (Dmitrieva & Ostroumov 1986), induce
courtship behavior (Ostroumov & Dmitrieva 1990) and synchronize
the release of sperm and eggs (Stacey & Hoursten 1982). The sex
pheromones of goldfish, Carassius auratus, currently are the most
extensively studied among those found in fish. Female goldfish
release 17,20 B-dihydroxy—4-pregnen-3-one and 17,200-dihydroxy-4-
pregnen-3—one sulfate into water just before ovulation (Stacey et
al. 1989; Sorensen et a1. 1995). Both substances are highly
stimulatory to the olfactory organ of conspecifics. Also, male
goldfish show elevated levels of plasma gonadotropin II (Dulka et
a1. 1987; Sorensen et a1. 1995) and sperm (Stacey et a1. 1989) in
response to these substances. In addition, post-ovulatory female
goldfish release prostaglandin F2d and its metabolites into
water, which also induce neuro—endocrine and behavioral responses
in male goldfish (Sorensen et a1. 1988, 1989). Male sex
pheromones, although less extensively studied in fish, have been
reported in numerous species such as Ictalurus catfish (Todd et

a1. 1967; Richards 1974); rainbow trout, Oncorhynchus mykiss

(Newcombe & Hartman 1973); blenny, Blennius pavo (Laumen et al.
1974); five belontiid species (Lee & Ingersoll 1979); black goby,
Gobius jozo (Colombo et a1. 1980, 1982); and Pacific herring,
Clupea harengus pallasi (Stacey & Hourston 1982; Sherwood et a1.
1991; Carolsfeld et al. 1997). These olfactory Signals have been
found to attract mature females to mature males or to induce
physiological changes such as ovulation in female conspecifics.

It has been suggested that sea lampreys, one of the lone
survivors of the most ancient vertebrate classes Agnatha, also
use male pheromones to coordinate spawning (Teeter 1980; Li
1994). Behavioral observations of sea lampreys suggest that
odorants released by adult males attract adult females and repel
other adult males (Teeter 1980; Li 1994). These findings are
consistent with the reproductive biology of the sea lamprey where
males usually initiate nest building and are later joined by one
or more females (Applegate 1950). Also, electro-olfactograms
made using female sea lampreys showed that washings from
spermiating males were a thousand times more potent than those
from non—spermiating males (Li 1994; Bjerselius et al. 1996).
However, there has been no direct link between this potent
spermiating male odorant and the attraction/repulsion behavioral
responses previously observed.

I formulated the hypothesis that spermiating male sea
lampreys release a potent and specific sex pheromone. Because
there has been no direct evidence of the significance of

spermiation to male pheromone release, experimental exploration

of my hypothesis should be focused on the timing of release and
specific behavioral mechanism of this pheromone. To test this
hypothesis I examined the following specific questions; 1) when
placed in a two-choice maze, whether post—ovulatory female sea
lampreys will be attracted or spend more time in the side of the
maze containing a spermiating male odorant, 2) when in the two—
choice maze, whether post—ovulatory female sea lampreys will show
increased searching behavior in the side of the maze containing a
spermiating male odorant, and 3) when observed in a natural
spawning stream exposed to odorants from both spermiating and
non—spermiating males, whether post-ovulatory females will be

attracted to the spermiating male odorant.

METHODS

Collection and Maintenance of Animals

Adult sea lampreys were trapped or collected by hand from
tributaries to Lakes Huron and Michigan of North America by the
staff of the United States Fish and Wildlife Service, Marquette
Biological Station, Marquette, Michigan USA. All adult sea
lampreys were transferred to the United States Geological Survey,
Hammond Bay Biological Station (HBBS), Presque Isle County,
Michigan USA. The sea lampreys were held in flow—through tanks

(1000L) with Lake Huron water at temperatures ranging from of 7°C
to 20°C.

Adult males were induced to spermiate by holding them at
18 oC and then injecting them intra—peritoneally with [D—Ala6]—LH-
RH(pGlu-His—Trp—Ser-Tyr—D-Ala-Leu-Arg-Pro—Gly-NHZ, Sigma Chemical

Co., St Louis, MO USA) at a dose of 100 ug/Kg body weight (Li

1994). This injection was repeated in two days and then again in
five days. Males were checked each day until spermiation
occurred. Females were Similarly treated to induce ovulation and

were checked each day until ovulation occurred.

Adult males and females were used as both test subjects and
odor sources for behavioral tests. Males were identified by a
raised dorsal ridge and females by an enlarged and soft abdomen
along with no dorsal ridge (Vladykov 1949). Each sex was further

assigned one of two maturity classifications. For males,

individuals that did not emit sperm after gentle pressure was
applied to the abdomen were classified as non—spermiating males
(NSM); males that did emit sperm were classified as spermiating
males (SM). For females, individuals that did not release eggs
after gentle pressure was applied to the abdomen were classified
as pre—ovulatory females (NOF); females that did release eggs
were classified as post-ovulatory females. These four
classifications formed the test subject and odor source

combinations used in experiments.

Behavioral Preference Tests

Preference tests were used to determine the attraction or
repulsion of adult sea lampreys within each sex and maturity
class to odorants produced by conspecific adults of each sex and
maturity class. These tests were carried out using a two—choice
(Y-maze) design (Figure 1). Water was able to flow through these
mazes, but sea lampreys were blocked from escaping by sealing the
maze with plastic mesh. Odor chambers are located at the head of
each side of the mazes. Two mazes were assembled in the raceways
at HBBS (referred to as indoor mazes). Another maze was
constructed at a field site located on the Ocqueoc River, Presque
Isle County, Michigan USA, a Lake Huron tributary (referred to as
outdoor maze, Figure 2) to independently confirm responses to
odorants in a different setting using river water. The Ocqueoc

River is a historic sea lamprey spawning stream (Applegate 1950)

4.27m 0.30m 0.61m 0.91m
I | | |

I
Z ‘— 1 3 :
1. 22m A“ J E l +_Water
' E S I
‘— // '
: 5 I

6.1m y

Odor Chambers

 

Figure 1. Design of indoor and outdoor two-choice mazes used in
preference and searching behavior tests. Arrows indicate water

flow. Dotted lines indicate flow through plastic mesh. Dashed

line indicates low—head laminar flow device.

 

Figure 2. Two—choice maze used in outdoor experiments. Water
was pumped into the head of the maze and allowed to flow through
back into the river. A single sea lamprey was introduced and its
behavior in the maze was monitored both before and after an
odorant from sea lampreys of a specific sex and maturity was
introduced.

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 low background levels
of odorants from other sea lampreys to interfere with behavioral
tests and yet offered water temperature and quality known to be
suitable for sea lamprey reproduction. The dimension of each arm
of the maze (see Figure 1) and the water flow (0.05 m/s indoor
and 0.07 m/s outdoor) were identical.

In all, numerous tests were conducted representing all
combinations of sex and maturity classes as test subjects and
odor sources. A single sea lamprey of a specific sex and
maturity class was chosen and introduced into the maze. The sea
lamprey was allowed to acclimate for 10 minutes and then its
behavior was video—recorded for 20 minutes. Then, sea lampreys
or washings from sea lampreys of a specific sex and maturity
class were introduced into the odor chamber of one side of the
maze chosen randomly by the toss of a coin. Actual fish (five
individuals used per test) or water in which spermiating males
were held (referred to as washings) were used for odor sources.
The odorant was allowed to perfuse through the maze for 5
minutes, and the behavior of the sea lamprey was recorded again
in the presence of the odor source for 20 minutes. The maze was
allowed to flush free of odorants for 10 minutes before another
test was started. Each test cycle lasted about 65 minutes from

acclimation to the end. Tests were conducted between 0700 and

1700 hours in water temperatures that ranged from 7°C to 20°C for

indoor tests and 12°C to 24°C for outdoor tests. Videotapes were
viewed and the time (seconds) spent in the treatment (odorant)
side and the control (no odorant) side before odorant
introduction (Berand Bc, where e is treatment and c is control)
and the time spent in each side after odorant introduction (Ae
and Ac, where e is treatment and c is control) were recorded.
Proportions of time spent in each side of the maze before and
after the odorant introduction (Ae/Be & Ac/Bc) were used to
calculate an index of preference (I)for each individual test

subject:

I = (Ae/Be) — (Ac/BC)

This index standardizes results by comparing test subject
behavior in each arm before and after odorant introduction. A
positive I value indicates an overall attraction and a negative
value an overall repulsion for a single test (see Appendices A
and B).

Tests in which sea lampreys did not explore both sides of
the maze in the 20 minutes before odorant introduction, and those
that did not explore either side in the 20 minutes after odorant
introduction, were not used in analysis. This was necessary
because proportions could not be generated from these situations
which occurred in approximately 25% of all tests. We attempted
to collect a sample size of 12 valid tests for indoor tests and

eight for outdoor tests for each combination of test subject and

odor source class. The I values (differences in the proportion
of times spent in the treatment and control sides of the maze)
were analyzed with a two—tailed Wilcoxon Signed Ranks Test (Rao
1998) to determine if there was significant attraction or
repulsion in each combination of test subject and odor source

class.

Searching Behavior Tests

The same two—choice mazes were used to assess the effect of
male odorants on searching behavior of adult sea lampreys in each
sex and maturity class. In the initial preference tests I
observed individual test subjects often displaying increased
swimming activity toward the mesh that was preventing them from
reaching the odor source at the head of the maze. This activity
is characterized by pacing back and forth across the upstream
block, increased swimming intensity and the rapid beating or
vibration of the tail as the sea lamprey tries to overcome the
upstream block. These activities were interpreted as searching
behavior. I reviewed the videotapes of preference tests where
spermiating males were the odor source (including spermiating
male washings) and also those where non—spermiating males were
the odor source and post-ovulatory females were the test
subjects. I recorded the time (seconds) spent searching in both
sides of the maze before and after the odorant introduction and

described the results similarly to those of the preference

-10-

experiments. Again, tests in which sea lampreys did not display
searching behavior in both Sides of the maze in the 20 minutes
before odorant introduction, and those that did not search in
either side in the 20 minutes after odorant introduction, were
not used in analysis. To promote objectivity in estimating the
time spent searching, the identity the treatment side was
withheld until after viewing. The indices of searching (see
Appendices C and D) were calculated similarly to the indices of
preference and analyzed with a Wilcoxon Signed Ranks Test to
determine whether the spermiating male odorant induces an

increase in searching activity.

Behavior in a Spawning Stream

To assess whether females exhibit similar preference and
activity behavior in a natural setting, I monitored pre and post-
ovulatory female behavior in response to male odorants in a 65
meter section of the Ocqueoc River, which was chosen for the
reasons stated earlier. At the upstream portion of the study
section, an island naturally divides the river (Figure 3). Cages
(1 m3) for odor sources were constructed with a wood frame
encased in plastic mesh (~1.5 cm gap) and were placed in the
channels on each side of the island. A block net (~1.5 cm gap
mesh) was placed at the downstream end of the section. An

acclimation cage (0.5 UP) for test subjects constructed with the

-11-

 

Figure 3. The upstream portion of the study section including an
island that divides the river and the odor cages used in spawning
stream behavior experiments. Post—ovulatory and pre—ovulated
female sea lampreys were fitted with radio tags and their
behavior monitored in response to spermiating and non—spermiating
male odorants.

_12_

same material was placed at the downstream end just above the
block net.

Tests were conducted between 0700 and 1700 hours in water
temperatures ranging from 12°C to 24°C. A day in advance of
testing a female sea lamprey (post—ovulatory or pre-ovulatory)
was fitted with a radio tag designed for external mount (Advanced
Telemetry System, Isanti, Minnesota USA). The following morning
sea lampreys being used for odor sources (spermiating males and
non—spermiating males) and the tagged test subject were
transported to the study site. The odor sources (spermiating and
non-spermiating males) were randomly assigned to the two cages on
the side of the island. The test subject could choose to swim to
spermiating males, non—spermiating males or to none at all. The
tagged female was then placed in the acclimation cage and allowed
to acclimate, exposed to the two odor sources, for 2 hours.

In each test the subject was released and its location
observed visually or by a radio receiver (Lotek Engineering Inc.,
Newmarket, Ontario, Canada) and recorded on a map grid of the
site every 5 minutes. If test subjects failed to move from the
release site within an hour they were removed. If tests subjects
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 day
(which usually was 4 hours from the start of the test).

A contingency table was used to tally the behavior of

female test subjects of different maturation. Categories of

-13-

response consisted of swimming to the spermiating male odor
source, swimming to the non-spermiating male odor source, or not
choosing an odor source (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
in a natural environment (proc freq, 1998 SAS Institute Inc.,
Cary, North Carolina USA). The times in which females swam to the

odor sources were also recorded (see Appendix E).

-14-

RESULTS

Behavioral Preference Tests

The behavior of sea lampreys observed in the two-choice
maze preference experiments varied greatly. Most sea lampreys
alternated between attachment to the side of the mazes and
swimming throughout the mazes. Sea lampreys showed no bias to
either side of the mazes.

The indoor two—choice mazes were used to characterize the
preference responses of individuals in all four sex and maturity
classes to all four sex and maturity classes of odor sources.
Post-ovulatory female preference responses to spermiating male
washings were also characterized. These experiments resulted in
17 combinations in all (Table 1). In most combinations, there
were not significant preference or avoidance responses (Wilcoxon
Signed Ranks Test, see Table l for sample sizes, P>O.10). The
most consistent preference observed was in post—ovulatory females
in response to a spermiating male odorant; 14 of 14 post—
ovulatory females spent significantly more time in the treatment
side when actual sea lampreys were used as the odor source
(Wilcoxon Signed Ranks Test, N=14, P<0.0001). In addition, 10 of
11 post—ovulatory females tested Spent significantly more time in
the control side of the maze (no odorant) when exposed to a non—

spermiating male odorant (Wilcoxon Signed Ranks Test, N=11,

-15-

Table 1. Number of adult sea lampreys of specific sex and
maturity choosing the treatment (odorant) or control (no odorant)
side of an indoor two-choice maze. The treatment side used
spermiating male (SM), non-spermiating male (NSM), post-ovulatory
female (OF), and pre-ovulatory female (NOF) sea lampreys and also
water collected from spermiating males (SMW) as odor sources.
Test subjects were spermiating males (SM), non-spermiating males
(NSM), post-ovulatory females (OF), and pre-ovulatory females
(NOF). Statistical significance was determined with a Wilcoxon
Signed Ranks Test (2-tailed) using indices of preference (see
Appendix A).

Odor Test Side Test
Source Subject n Treatment Control Statistic p value

 

 

SM OF 14 14 0 105 <0.0001
SM NOF 16 4 12 34 NS
SM SM 12 6 6 49 NS
SM NSM ll 7 4 36 NS
SMW OF 8 6 2 32 NS
NSM OF 11 l 10 1 0.002
NSM NOF 29 12 17 173 NS
NSM SM 18 8 10 76 NS
NSM NSM 1d. 4 7 31 NS
OF OF 14 8 6 84 NS
OF NOF 11 7 4 37 NS
OF SM 18 15 3 135 NS
OF NSM 11 5 6 41 NS
NOF OF 13 6 7 35 NS

 

~16-

 

 

Table l (cont’d).
Odor Test Side Test
Source: Subject n Treatment Control Statistic p value
NOF NOF l l 5 6 3 7 NS
NOF SM 13 8 5 4 9 NS
NOF NSM l l 4 7 24 NS

 

-17-

P=0.002). Further, 6 of 8 post-ovulatory females spent more time
in the side of the maze with a spermiating male odorant when
washings were used as an odor source even though the statistical
outcome was not significant.

The outdoor two—choice maze was used to further explore the
preference responses to a spermiating male odorant observed in
the inside mazes (Table 2). These tests showed that 8 of 8 post-
ovulatory female sea lampreys spent significantly more time in
the treatment side of the maze when actual spermiating males were
the odor source (Wilcoxon Signed Ranks Test, N=8, P=0.004).

Also, 7 of 8 post-ovulatory females tested spent more time in the
side of the maze containing a spermiating male odorant when
washings were used as the odor source even though the outcome was
not significant. Individuals in all other sex and maturity
classes did not show this same behavior towards a spermiating
male odorant. Indices of preference for indoor and outdoor

experiments are displayed in Appendices A and B.

Searching Behavior Tests

Post—ovulatory female sea lampreys in both indoor and
outdoor experiments increased their searching activity when
exposed to a spermiating male odorant (Table 3 and Table 4).
When actual spermiating males were used as an odor source, 11 of

11 (indoor mazes, Wilcoxon Signed Ranks Test, N=11, P<0.0001) and

-13-

Table 2. Number of adult sea lampreys of specific sex and
maturity choosing the treatment (odorant) or control (no odorant)
side of an outdoor two—choice maze. The treatment side used
spermiating male (SM) and non—spermiating male (NSM) sea lampreys
and also water collected from spermiating males (SMW) as odor
sources. Test subjects were spermiating males (SM), non-
spermiating males (NSM), post-ovulatory females (OF), and pre-
ovulatory females (NOF). Statistical significance was determined
with a Wilcoxon Signed Ranks Test (2-tailed) using indices of
preference (see Appendix B).

Odor Test Side Test
Source: Subject n Treatment Control Statistic p value

 

 

SM OF 8 8 0 36 0.004
SM NOF 8 S 3 17 NS
SM SM 10 6 4 30 NS
SM NSM 13 6 7 36 NS
SMW OF 8 7 1 33 NS
NSM OF 10 7 3 41 NS

 

-19-

Table 3. Number of adult sea lampreys of Specific sex and
maturity spending more time searching in the treatment (odorant)
or control (no odorant) side of an indoor two-choice maze. The
treatment side used spermiating male (SM) and non—spermiating
male (NSM) sea lampreys and also water collected from spermiating
males (SMW) as odor sources. Test subjects were spermiating
males (SM), non-spermiating males (NSM), post-ovulatory females
(OF), and pre-ovulatory females (NOF). Statistical significance
was determined with a Wilcoxon Signed Ranks Test (2—tailed) using
indices of searching (see Appendix C).

Odor Test Side Test
Source: Subject n Treatment Control Statistic P-value

 

 

SM OF 11 ll 0 66 <0.0001
SM NOF 13 7 6 46 NS
SM SM 9 4 5 21 NS
SM NSM 10 6 4 33 NS
SMW OF 7 7 0 29 0 . 008
NSM OF 9 4 5 19 NS

 

-20-

Table 4. Number of adult sea lampreys of specific sex and
maturity spending more time searching in the treatment (odorant)
or control (no odorant) side of an outdoor two-choice maze. The
treatment side used spermiating male (SM) and non—spermiating
male (NSM) sea lampreys and also water collected from spermiating
males (SMW) as odor sources. Test subjects were spermiating
males (SM), non-spermiating males (NSM), post-ovulatory females
(OF), and pre-ovulatory females (NOF). Statistical significance
was determined with a Wilcoxon Signed Ranks Test (2-tailed) using
indices of searching (see Appendix D).

Odor Test Side Test
Source: Subject n Treatment Control Statistic P-value

 

 

SM OF 8 8 0 36 0 . 004
SM NOF 6 3 3 13 NS
SM SM 10 6 4 39 NS
SM NSM 8 2 6 13 NS
SMW OF 7 7 0 29 0 . 008
NSM OF 7 4 3 22 NS

 

-21-

8 of 8 (outdoor mazes, Wilcoxon Signed Ranks Test, N=8, P=0.004)
post-ovulatory females tested showed a significant increase in
searching behavior in the side of the maze with this odor source.
Also, when washings from spermiating males were used as an odor
source 7 of 7 post—ovulatory females tested (both inside and
outside mazes, Wilcoxon Signed Ranks Test, N=7, P=0.008) showed a
significant increase in searching behavior in the side containing
the odorant. Individuals in all other sex and maturity classes
did not show significant increases in searching behavior in
response to a spermiating male odorant. Indices of searching for
indoor and outdoor experiments are displayed in Appendices C and

D.

Behavior in a Spawning Stream

The odorant released by spermiating males appeared to
influence the behavior of post-ovulatory females within the
spawning stream section (Table 5). The behavior of adult female
sea lampreys in the Ocqueoc River varied greatly, ranging from
fast movement upstream towards one of the odor cages to no
movement from the acclimation cage. Females were not biased to
either side of the stream. Of 13 post-ovulatory females tested,
nine swam to and then stayed at the cage containing spermiating
males, two stayed at an intermediate position in the study area,
and two did not move upstream from the acclimation cage. No

post—ovulatory females swam to the cage containing

-22-

Table 5. Number of post-ovulatory (OF) and pre-ovulatory (NOF)
female sea lampreys swimming to spermiating males (SM), non-
spermiating males (NSM), or staying at an intermediate (Int.)
position within a section of a known sea lamprey spawning stream.
The distribution of choices were significantly different between
post—ovulatory and pre-ovulatory females (Fisher's Exact Test, 2-
Tail, P = 0.024).

Behavioral Choice

 

Maturation SM NSM Int.
OF 9 O 4
NOF 1 2 4

 

-23-

non-spermiating males. Among the seven pre—ovulatory females
tested, one swam to the cage containing spermiating males, two
swam to the cage containing non-spermiating males, three stayed
at an intermediate position in the study area, and one did not
move upstream from the acclimation cage. The distribution of
choices was significantly different between pre and post-
ovulatory females suggesting the attraction of post—ovulatory
females to spermiating males (Fisher’s Exact Test P=0.024). The
actual times in which females reached the odor sources are

displayed in Appendix E.

-24-

DISCUSSION

All three experiments support my hypothesis that the odorant
released by spermiating male sea lampreys is a sex pheromone. In
the first experiment where preference/avoidance responses were
observed, post-ovulatory females spent more time in the side of
the maze with a spermiating male odorant. In contrast, post-
ovulatory females did not show a similar preference for the
odorants of either non—spermiating males or other female sea
lampreys (pre and post-ovulatory). This strict specificity in
the sex and maturity of the releaser and receiver of the signal
suggests it does not function in general aggregation. Also, this
signal seems to function through chemoreception, most likely
olfaction because a similar preference response was observed from
post-ovulatory females when using washings from spermiating
males, which eliminated detection by certain other sensory modes
such as sight, sound and electricity. The most likely function
of an odorant with this specificity is as a sex pheromone
coordinating mating activity.

The attraction response of post-ovulatory female sea
lampreys appears to be mediated through an increase in searching
behavior. In the second experiment post—ovulatory females swam
more actively upstream in the side of the maze with a spermiating
male odorant. This behavior makes sense because it would bring
them to the odor source (ripe males in a spawning stream). This

potential is confirmed in the third experiment in which post—

-25-

ovulatory females are found to localize spermiating males in an
actual spawning stream. This also confirms that the odorant is
potent enough to function in their natural habitat. Taken
together, my three lines of behavioral experiments demonstrate
that this odorant is a specific and potent sex pheromone.

The hypothesis that spermiating male sea lampreys release a
sex pheromone is also supported by previous observations. French
fishermen have been known to bait their traps with adult male sea
lampreys in hopes of increasing their catch rates (Hardisty &
Potter 1971). Past behavioral studies suggested that adult male
sea lampreys produce an odorant that not only attracts adult
females, but also repels other adult males (Teeter 1980; Li
1994). My results showed that males release a pheromonal odorant
only during spermiation and that it specifically attracts post-
ovulatory females. These varying results could be explained by
differences in classification of sexual maturity in both males
and females. What is notable, is that in all previous studies
and in my results, an attraction of some degree to a male odorant
was observed in females. My study extends this knowledge by
showing the significance of maturation (spermiation and
ovulation) in the function of this sex pheromone system to
attract only post-ovulatory females to spermiating males.

Reproductive biology of the sea lamprey also suggests that
a male sex pheromone system would be critical to coordination of
spawning behavior. Mating begins with nest construction that is

typically initiated by a male who is then joined by females

-26-

(Applegate 1950). Sea lampreys die shortly after ovulation and
spermiation (Applegate & Smith 1951; Larsen 1980), so they only
have a limited period of time to reproduce. An attractant would
be effective in serving as a final link in synchronization of
spawning allowing males to draw ripe females to their nests and
also for females to locate males that are ready to spawn.

It has been demonstrated that a wide variety of post—larval
sea lamprey behaviors are regulated by odorants. Studies Show
that the olfactory system is well—developed (Kleerekoper 1972)
and important in prey searching and migration. Anatomical
studies show that the olfactory organ is relatively large and its
sensory epithelium is further enlarged through longitudinal
folding (Kleerekoper & van Erkel 1960; Thornhill 1967). Post—
larval lampreys have the largest proportion of the brain
dedicated to the olfactory sense among vertebrates (Stoddart
1990). Prey searching experiments have shown that parasitic sea
lampreys, when placed in a two choice maze, show greater swimming
activity and are attracted to rinses from brook trout, Salvelinus
fontinalis, (Kleerekoper & Mogensen 1959; 1963) which are
potential prey. These responses are eliminated by blocking the
nose (Kleerekoper 1972). Migratory pheromone studies have also
Shown that adults are attracted to conspecific larvae (Teeter
1980; Li 1994). Adults are found to be sensitive to a unique
bile acid, petromyzonol sulfate, released by larval sea lampreys
(Li et al. 1995). It appears that the post—larval life stage of

the sea lamprey relies on olfaction for prey searching and

-27-

migration. My study now also documents the use of olfaction for
mating.

In summary, it is evident that spermiating male sea
lampreys release a potent sex pheromone that specifically induces
an increase in searching behavior, which ultimately brings post—
ovulatory females to spermiating males. My results, combined
with previous electrophysiology studies, clearly demonstrate the
significance of spermiation and ovulation in the release of and
response to this pheromone. More research is needed to clarify
the exact pattern of release and the physiological mechanism for

the production of this pheromone.

-28-

APPENDICES

-29-

APPENDIX A

Indoor Preference Test Raw Data

-30-

Table 6. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for post-ovulatory female test subjects exposed to a
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two—tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of

Trial Before .After Before .After' Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)

l 58 4 463 209 0.3824

2 204 145 504 561 0.4023

3 476 333 362 464 0.5822

4 130 69 188 229 0.6873

5 149 57 139 204 1.0851

6 227 71 475 808 1.3883

7 230 63 254 561 1.9347

8 260 74 136 328 2.1271

9 356 0 104 341 3.2788

10 226 0 289 1200 4.1522

11 429 0 95 433 4.5579

12 85 0 250 1200 4.8000

13 127 79 141 951 6.1226

14 61 38 62 600 9.0545

 

-31-

Table 7. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for pre-ovulatory female test subjects exposed to a
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After' Before .After‘ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 72 9 132 15 -0.0114
2 360 379 465 479 -0.0227
3 365 418 350 301 -0.2852
4 174 40 84 47 0.3296
5 191 130 307 30 -0.5829
6 347 582 269 121 —1.2274
7 260 0 180 239 1.3278
8 232 564 357 252 -1.7252
9 282 580 491 104 -1.8449
10 143 88 185 484 2.0008
11 242 535 491 50 -2.1089
12 169 484 573 228 —2.4660
13 373 162 174 544 2.6921
14 86 241 117 0 —2.8023
15 123 508 571 113 —3.9322
16 93 586 576 77 -6.1674

 

-32-

Table 8.

Time spent (seconds) in each side of the indoor mazes

before and after odorant introduction and indices of preference
(I) for spermiating male test subjects exposed to a spermiating

male odorant.

The absolute values of

Positive I values indicate an attraction and
negative I values indicate a repulsion.

the I values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

 

 

 

Control Treatment Index of
Trial Before .After* Before .After' Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 269 89 308 92 -0.0322
2 287 390 438 496 -0.2265
3 29 47 215 276 -0.3370
4 449 112 114 123 0.8295
5 80 0 870 957 1.1000
6 365 438 314 15 -1.1522
7 84 0 891 1096 1.2301
8 220 390 343 167 -1.2858
9 280 1026 80 152 -1.7643
10 421 0 448 1000 2.2321
11 401 82 52 382 7.1417
12 120 63 46 685 14.3663

 

-33-

Table 9. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for non-spermiating male test subjects exposed to a
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 97 123 512 575 -0.l450
2 222 191 454 563 0.3797
3 199 293 461 432 -0.5353
4 249 209 396 546 0.5394
5 393 126 377 523 1.0667
6 215 362 351 156 -1.2393
7 550 41 377 548 1.3790
8 76 69 205 547 1.7604
9 318 582 321 0 1.8302
10 310 97 54 140 -2.2797
11 56 407 338 454 5.9247

 

-34-

Table 10. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for post-ovulatory female test subjects exposed to a
spermiating male washing odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 272 220 150 37 -0.5622
2 128 39 311 319 0.7210
3 296 427 539 7 —1.4296
4 326 47 162 371 2.1460
5 131 0 555 1200 2.1622
6 337 62 385 983 2.3693
7 195 31 104 279 2.5237
8 293 9 182 886 4.8374

 

-35-

Table 11. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for post-ovulatory female test subjects exposed to a non—
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After' Before .After“ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 447 71 24 6 0.0912
2 253 74 404 26 —0.2281
3 47 47 260 194 —0.2538
4 183 86 56 0 -0 4699
5 127 224 295 332 -0.6384
6 151 110 43 0 —0.7285
7 373 462 583 286 -0.7480
8 164 271 349 257 ~0.9160
9 123 305 175 216 -1.2454
10 67 390 156 74 —5.3465
11 18 195 622 606 -9.8591

 

-36-

Table 12. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for pre—ovulatory female test subjects exposed to a non-
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two—tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After’ Before .After~ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 408 335 360 283 -0.0350
2 257 281 195 184 -0.1498
3 441 446 265 333 0.2453
4 174 175 545 713 0.3025
5 360 226 493 473 0.3317
6 238 136 209 49 -0.3370
7 297 274 392 502 0.3581
8 154 219 286 574 0.5849
9 387 425 251 122 —0.6121
10 278 226 385 63 —0.6493
11 358 493 411 220 -0.8418
12 238 192 346 606 0.9447
13 307 445 255 118 —0.9868
14 509 559 40 1 —1.0732
15 156 311 469 403 -1.1343
16 182 0 467 533 1.1413

 

-37-

Table 12 (cont’d).

 

 

 

Control Treatment Index of
Trial Before After Before .After* Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
17 411 659 266 115 -1.1711
18 559 167 261 410 1.2721
19 481 337 168 355 1.4125
20 197 444 250 207 -1.4258
21 174 409 507 464 —1.4354
22 373 649 389 0 -1.7399
23 116 295 503 305 —1.9367
24 128 348 551 419 —1.9583
25 111 323 240 207 -2.0474
26 221 55 82 249 2.7877
27 128 0 394 1200 3.0457
28 606 0 260 977 3.7577
29 22 217 462 339 —9.1299

 

-38-

Table 13. Time Spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for spermiating male test subjects exposed to a non-
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed using a Wilcoxon
Signed Ranks Test (two-tailed)to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After‘ Before .After' Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 224 187 187 153 -0.0166
2 889 30 140 0 —0.0337
3 42 69 75 116 -0.0962
4 304 30 138 27 .0970
5 236 165 387 312 .1070
6 216 0 152 29 .1908
7 228 220 214 250 .2033
8 210 92 63 4 -0.3746
9 215 560 88 187 —0.4797
10 294 337 166 85 -0.6342
11 319 26 103 82 0.7146
12 146 47 259 282 0.7669
13 28 73 31 44 -1.1878
14 33 67 682 495 —l.3045
15 334 O 625 1200 1.9200
16 515 361 121 333 2.0511

 

-39-

 

 

 

Table 13 (cont’d).
Control Treatment Index of
Trial Before .After" Before .After" Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
17 532 1200 123 0 -2.2556
18 24 76 338 137 —2.7613

 

-40-

Table 14. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for non-spermiating male test subjects exposed to a non—
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two—tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before After Before .After' Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 125 101 689 468 —0.1288
2 269 166 417 188 -0.1663
3 175 48 468 268 0.2984
4 384 316 320 136 -0.3979
5 438 351 158 59 -0.4280
6 362 368 492 274 -0.4597
7 297 348 216 398 0.6709
8 83 172 716 558 -l.2930
9 81 206 509 575 -1.4135
10 326 154 118 348 2.4768
11 560 437 22 133 5.2651

 

-41-

Table 15. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for post-ovulatory female test subjects exposed to a post—
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After‘ Before .After‘ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 800 412 282 144 —0.0044
2 37 57 359 549 -0.0113
3 165 136 365 268 -0.0900
4 328 246 359 183 -0.2403
5 244 378 162 183 -0.4196
6 43 72 768 934 -O.4583
7 678 148 205 145 0.4890
8 216 51 361 299 0.5921
9 419 182 751 958 0.8413
10 566 442 216 630 2.1357
11 267 13 93 374 3.9728
12 303 38 43 246 5.5955
13 372 26 104 600 -5.6993
14 133 0 72 1200 —16.6667

 

-42-

Table 16. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for pre—ovulatory female test subjects exposed to a post—
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 113 116 613 706 0.1252
2 272 216 188 232 0.4399
3 434 444 88 41 -0.5571
4 434 205 392 430 0.6246
5 512 677 229 147 -0.6803
6 75 114 214 493 0.7837
7 510 345 193 319 0.9764
8 432 46 456 755 1.5492
9 122 229 120 416 1.5896
10 42 126 316 217 —2.3133
11 26 118 97 210 -2.3735

 

-43-

Table 17. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for spermiating male test subjects exposed to a post-
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two—tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 150 93 230 196 0.2322
2 277 0 137 35 0.2555
3 347 152 345 368 0.6286
4 265 98 260 272 0.6763
5 340 330 275 509 0.8803
6 381 129 395 500 0.9272
7 136 192 119 51 -0.9832
8 18 55 10 41 1.0444
9 115 116 136 288 1.1090
10 250 235 94 223 1.4323
11 65 0 169 280 1.6568
12 558 170 248 527 1.8203
13 329 148 205 472 1.8526
14 22 70 97 112 —2.0272
15 428 1200 124 0 -2.8037
16 109 100 56 225 3.1004

 

-44-

 

 

 

Table 17 (cont’d).
Control Treatment Index of
Trial Before After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
17 50 0 193 748 3.8756
18 69 74 28 271 8.6061

 

-45_

Table 18. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for non-spermiating male test subjects exposed to a post-
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After‘ Before .After" Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 188 254 288 383 —0.0212
2 189 195 428 419 -0.0528
3 319 436 290 155 -0.8323
4 259 235 298 10 -0.8738
5 474 588 240 85 -0.8863
6 172 0 364 402 1.1044
7 323 152 412 667 1.1483
8 468 100 320 488 1.3113
9 71 0 419 666 1.5895
10 325 596 318 30 -1.7395
11 381 179 16 266 16.1552

 

-46-

Table 19. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for post-ovulatory female test subjects exposed to a pre-
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After* Before .After' Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 271 232 231 205 0.0314
2 152 238 267 427 0.0335
3 166 25 135 0 -0.1506
4 233 221 198 225 0.1879
5 433 494 313 290 -0.2144
6 138 54 378 345 0.5214
7 299 377 722 352 —0.7733
8 309 577 258 227 -0.9875
9 151 357 279 364 -1.0596
10 271 132 56 261 4.1736
11 29 195 685 898 -5.4132
12 95 35 7 144 20.2030
13 13 589 454 219 -44.8253

 

-47-

Table 20. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for pre-ovulatory female test subjects exposed to a pre-
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 236 29 47 0 —0.1229
2 202 152 90 45 -0.2525
3 167 50 54 0 -0.2994
4 79 72 273 332 0.3047
5 2 0 25 12 0.4800
6 249 272 435 237 -0.5475
7 71 105 279 219 -0.6939
8 84 41 243 309 0.7835
9 364 409 41 98 1.2666
10 102 255 328 243 —1.7591
11 182 31 170 448 2.4650

 

-48-

Table 21. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for spermiating male test subjects exposed to a pre-ovulatory
female odorant. Positive I values indicate an attraction and
negative I values indicate a repulsion. The absolute values of
the I values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

 

 

 

Control Treatment Index of
Trial Before After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 248 279 489 616 0.1347
2 454 426 319 252 -0.1484
3 207 143 358 317 0.1947
4 235 34 121 57 0.3264
5 154 75 330 289 0.3887
6 396 217 246 244 0.4439
7 109 50 123 134 0.6307
8 218 326 522 365 -0.7962
9 337 444 326 165 —0.8114
10 320 688 162 151 -1.2179
11 508 O 148 202 1.3649
12 92 0 658 1113 1.6915
13 303 814 550 220 —2.2865

 

-49-

Table 22. Time spent (seconds) in each side of the indoor mazes
before and after odorant introduction and indices of preference
(I) for non-spermiating male test subjects exposed to a pre—
ovulatory female odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After* Before .After“ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 282 226 544 369 —0.1231
2 529 460 237 166 -0.1691
3 388 433 139 71 -0.6052
4 483 369 272 380 0.6331
5 553 427 115 197 0.9409
6 243 399 421 191 —1.1883
7 299 3 716 877 1.2148
8 198 316 298 838 1.2161
9 83 187 126 117 -1.3244
10 292 472 392 110 -1.3358
11 235 410 351 135 —1.3601

 

-50-

APPENDIX B

Outdoor Preference Test Raw Data

-51-

Table 23. Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of preference
(I) for post—ovulatory female test subjects exposed to a
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After Before .After* Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)
1 472 358 370 343 0.1686
2 353 493 155 445 .4744
3 641 361 263 551 .5319
4 307 186 203 617 .4335
5 179 22 229 1115 .7461
6 369 53 144 835 .6550
7 158 52 28 838 29.5995
8 549 79 1 1013 1012.8561

 

-52-

Table 24. Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of preference
(I) for pre-ovulatory female test subjects exposed to a
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After' Before .After' iPreference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)

1 321 117 150 65 0.0688

2 216 251 162 237 0.3009

3 180 48 797 877 0.8337

4 612 868 41 121 1.5329

5 253 556 208 85 -1.7890

6 132 369 475 325 -2.1112

7 114 0 281 959 3.4128

8 54 298 210 413 -3.5519

 

-53-

Table 25. Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of preference
(I) for spermiating male test subjects exposed to a spermiating
male odorant. Positive I values indicate an attraction and
negative I values indicate a repulsion. The absolute values of
the I values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall preference behavior.

 

 

Control Treatment Index of
'Trial Before .After Before .After Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 476 149 313 55 -0.1373
2 188 148 169 216 0.4909
3 271 154 580 676 0.5973
4 341 399 275 502 0.6554
5 573 236 100 127 0.8581
6 171 0 457 413 0.9037
7 332 639 174 39 —1.7006
8 215 569 748 426 -2.0770
9 339 855 472 184 -2.1323
10 229 0 247 767 3.1053

 

-54-

 

Table 26. Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of preference
(I) for non—spermiating male test subjects exposed to a
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After Before .After“ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 431 478 552 595 —0.0312
2 504 407 321 271 0.0367
3 377 441 39 49 0.0866
4 188 190 120 147 0.2144
5 226 343 490 514 -0.4687
6 553 585 253 144 -0.4887
7 364 228 321 403 0.6291
8 25 0 1033 930 0.9003
9 229 501 411 329 —1.3873
10 130 246 339 87 —1.6357
11 211 472 457 141 —1.9284
12 472 490 76 410 4.3566
13 42 307 658 225 —6.9676

 

-55-

 

Table 27. Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of preference
(I) for post—ovulatory female test subjects exposed to a
spermiating male washing odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

Control Treatment Index of
Trial Before .After* Before .After“ Preference (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)

1 708 902 157 216 0.1018

2 621 548 76 102 0.4597

3 160 154 450 162 —0.6025

4 351 0 663 692 1.0437

5 225 0 540 921 1.7056

6 247 60 299 806 2.4527

7 260 3 186 573 3.0691

8 602 656 52 424 7.0641

 

-56-

Table 28. Time spent (seconds) in each side of the outdoor maze
before and after odorant introduction and indices of preference
(I) for post-ovulatory female test subjects exposed to a non-
spermiating male odorant. Positive I values indicate an
attraction and negative I values indicate a repulsion. The
absolute values of the I values were analyzed with a Wilcoxon
Signed Ranks Test (two-tailed) to determine overall preference
behavior.

 

 

 

Control Treatment Index of
Trial Before .After' Before .After' Preference (I)
Number (BC) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)

1 414 368 381 385 0.1216

2 327 251 613 244 -0.3695

3 439 177 138 0 -0.4032

4 56 35 339 501 0.8529

5 268 22 241 249 0.9511

6 486 0 638 1200 1.8809

7 855 241 341 776 1.9938

8 17 0 128 312 2.4375

9 219 711 612 217 -2.8920

10 396 145 40 723 17.7088

 

-57-

 

APPENDIX C

Indoor Searching Behavior Raw Data

-58-

Table 29. Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and indices of
searching (I) for post—ovulatory female test subjects exposed to
a spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two—tailed) to
determine overall searching behavior.

 

 

 

Control Treatment Index of
Trial Before After Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 129 85 151 200 0.6656
2 15 20 150 300 0.6667
3 44 12 67 86 1.0109
4 52 11 85 110 1.0826
5 325 200 110 215 1.3392
6 135 15 135 240 1.6667
7 75 30 180 465 2.1833
8 25 25 76 258 2.3947
9 450 45 30 75 2.4000
10 240 360 15 60 2.5000
11 24 17 15 480 31.2917

 

-59-

 

Table 30. Time spent (seconds) searching in each Side of the
indoor mazes before and after odorant introduction and indices of
searching (I) for pre-ovulatory female test subjects exposed to a
spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two—tailed) to
determine overall searching behavior.

 

 

Control Treatment Index of
Trial Before .After' Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be) - (Ac/Bc)
1 120 120 188 195 0.0372
2 68 15 30 15 0.2794
3 68 30 120 15 -0.3162
4 225 173 143 45 -0.4542
5 113 0 75 45 0.6000
6 98 165 135 120 —0.7948
7 90 38 105 135 0.8635
8 105 68 120 203 1.0440
9 105 255 135 98 -1.7026
10 113 210 158 15 -1.7635
11 173 90 60 195 2.7298
12 143 120 38 210 4.6872
13 38 218 225 38 -5.5680

 

~60-

Table 31. Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and indices of
searching (I) for spermiating male test subjects exposed to a
spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-tailed) to
determine overall searching behavior.

 

 

Control Treatment Index of
Trial Before .After' Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 83 15 83 0 —0.1807
2 45 75 128 180 -0.2604
3 195 83 113 135 0.7690
4 30 0 195 165 0.8462
5 105 90 83 0 -0.8571
6 15 0 113 128 1.1327
7 60 90 195 38 -1.3051
8 45 23 38 143 3.2520
9 30 210 23 0 ~7.0000

 

-61-

Table 32. Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and indices of
searching (I) for non-spermiating male test subjects exposed to a
spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-tailed) to
determine overall searching behavior.

 

 

 

Control Treatment Index of
Trial Before .After* Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
l 90 53 90 45 —0.0889
2 30 23 173 173 0.2333
3 60 0 90 38 0.4222
4 68 98 165 165 -0.4412
5 98 68 150 173 0.4595
6 75 60 173 218 0.4601
7 45 30 90 105 0.5000
8 68 113 195 165 -0.8156
9 158 218 120 30 -1.1297
10 158 15 128 210 1.5457

 

-62-

 

Table 33. Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and indices of
searching (I) for post—ovulatory female test subjects exposed to
a spermiating male washing odorant. Positive I values indicate
more searching in treatment side and negative I values indicate
more searching in control side. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test (two—
tailed) to determine overall searching behavior.

 

 

 

Control Treatment Index of
Trial Before .After Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 115 100 250 255 0.1504
2 200 175 150 180 0.3250
3 90 70 45 65 0.6667
4 300 150 275 400 0.9545
5 35 25 55 105 1.1948
6 135 100 35 95 1.9735
7 25 15 15 50 2.7333

 

-63-

Table 34. Time spent (seconds) searching in each side of the
indoor mazes before and after odorant introduction and indices of
searching (I) for post—ovulatory female test subjects exposed to
a non-spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-tailed) to
determine overall searching behavior.

 

 

Control Treatment Index of
Trial Before After Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)—(Ac/Bc)

l 15 15 45 30 -0.3333

2 60 15 38 23 0.3553

3 38 38 60 38 -0.3667

4 60 45 105 120 0.3929

5 23 15 105 120 0.4907

6 60 30 38 0 -0.5000

7 203 180 315 90 -0.6010

8 15 0 68 120 1.7647

9 68 195 150 23 -2.7143

 

-64-

APPENDIX D

Outdoor Searching Behavior Raw Data

-65-

Table 35. Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction and indices
of searching (I) for post—ovulatory female test subjects exposed
to a spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-tailed) to
determine overall searching behavior.

 

 

 

Control Treatment Index of
Trial Before .After' Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 170 146 146 212 0.5932
2 67 25 62 86 1.0140
3 110 89 56 148 1.8338
4 159 180 37 174 3.5706
5 104 0 72 405 5.6250
6 49 30 23 190 7.6486
7 25 26 3 527 174.6267

 

-66-

Table 36. Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction and indices
of searching (I) for pre-ovulatory female test subjects exposed
to a spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-tailed) to
determine overall searching behavior.

 

 

 

Control Treatment Index of
Trial Before .After' Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 127 211 48 45 -0.7239
2 78 181 253 146 —1.7434
3 103 23 223 537 2.1848
4 16 72 35 273 3.3000
5 18 75 414 287 -3.4734
6 17 0 31 808 26.0645

 

-67-

Table 37. Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction and indices
of searching (I) for spermiating male test subjects exposed to a
spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two-tailed) to
determine overall searching behavior.

 

 

 

Control Treatment Index of
Trial Before .After Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 122 86 132 87 -0.0458
2 166 50 88 14 —0.1421
3 89 42 41 27 0.1866
4 60 31 52 67 0.7718
5 157 122 94 146 0.7761
6 82 146 24 19 -0.9888
7 85 209 228 158 -1.7658
8 29 0 44 80 1.8182
9 36 0 35 77 2.2000
10 213 49 69 169 2.2192

 

-68-

Table 38. Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction and indices
of searching (I) for non-spermiating male test subjects exposed
to a spermiating male odorant. Positive I values indicate more
searching in treatment side and negative I values indicate more
searching in control Side. The absolute values of the I values
were analyzed with a Wilcoxon Signed Ranks Test (two—tailed) to
determine overall behavior.

 

 

 

Control Treatment Index of
Trial Before .After‘ Before .After Searching (I)
Number' (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 254 185 176 125 -0.0181
2 109 116 87 37 -0.6389
3 97 160 263 214 —0.8358
4 74 88 157 21 —1.0554
5 54 120 65 52 -1.4222
6 40 62 7 29 2.5929
7 128 78 35 131 3.1335
8 20 138 159 81 —6.3906

 

-69-

Table 39. Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction and indices
of searching (I) for post-ovulatory female test subjects exposed
to a spermiating male washing odorant. Positive I values
indicate more searching in treatment side and negative I values
indicate more searching in control side. The absolute values of
the I values were analyzed with a Wilcoxon Signed Ranks Test
(two-tailed) to determine overall searching behavior.

 

 

Control Treatment Index of
Trial Before .After Before .After Searching (I)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)
1 145 210 170 350 0.6105
2 25 14 15 55 3.1067
3 110 90 50 150 2.1818
4 200 150 195 300 0.7885
5 40 45 45 165 2.5417
6 15 10 7 45 5.7619
7 175 97 67 127 1.3412

 

_70-

Table 40. Time spent (seconds) searching in each side of the
outdoor mazes before and after odorant introduction and indices
of searching (I) for post—ovulatory female test subjects exposed
to a non-spermiating male odorant. Positive I values indicate
more searching in treatment side and negative I values indicate
more searching in control side. The absolute values of the I
values were analyzed with a Wilcoxon Signed Ranks Test (two—
tailed) to determine overall preference behavior.

 

 

 

Control Treatment Index of
Trial Before After Before .After Searching (1)
Number (Bc) (Ac) (Be) (Ae) (Ae/Be)-(Ac/Bc)

1 133 49 32 0 -0.3684

2 98 88 206 55 -0.6310

3 100 184 190 215 —0.7084

4 31 0 69 60 0.8696

5 40 25 38 57 0.8750

6 295 122 70 215 2.6579

7 25 20 36 184 4.3111

 

-71-

APPENDIX E

Spawning Stream Behavior Raw Data

-72-

 

Table 41. Post-ovulatory females choosing spermiating males
(SM), non-spermiating males (NSM) or staying at an intermediate
position (Int.) within a spawning stream study section. Times in
which females reached either spermiating or non—spermiating males
are recorded in minutes. Time is not applicable (NA) to those
females choosing an intermediate position.

 

Trial Time
Number Choice (minutes)
1 SM 25
2 SM 45
3 SM 10
4 SM 15
5 SM 15
6 SM 10
7 SM 45
8 SM 10
9 SM 15
10 Int . NA
11 Int . NA
12 Int . NA
13 Int . NA

 

-73-

Table 41. Pre-ovulatory females choosing spermiating males (SM),
non-spermiating males (NSM) or staying at an intermediate
position (Int.) within a spawning stream study section. Times in
which females reached either spermiating or non-spermiating males
are recorded in minutes. Time is not applicable (NA) to those
females choosing an intermediate position.

 

Trial Time
Number Choice (minutes)
1 NSM 10
2 NSM 15
3 SM 8 5
4 Int. NA
5 Int . NA
6 Int. NA
7 Int . NA

 

-74-

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-75-

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