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INVESTIGATING POTENTIAL APPLICATIONS OF THE MALE
MATlNG PHEROMONE IN SEA LAMPREY MANAGEMENT
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2/05 c:/ClFIC/DateDue.indd-p.15

INVESTIGATING POTENTIAL APPLICATIONS OF THE MALE MATING
PHEROMONE IN SEA LAMPREY MANAGEMENT

By

Nicholas S. Johnson

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

MASTER OF SCIECNE
Department of Fisheries and Wildlife

2005

ABSTRACT

INVESTIGATING POTENTIAL APPLICATIONS OF THE MALE MATING
PHEROMONE IN SEA LAMPREY MANAGEMENT

By
Nicholas S. Johnson

Spermiating male sea lampreys (Petromyzon marinas) release mating pheromones
that are highly attractive to ovulating females and influence their locomotion in spawning
streams. It has been hypothesized that pheromone-baited traps might be used to directly
remove females from spawning grounds and aid sea lamprey management in the Great
Lakes. However, no field studies have been conducted to determine the efficacy of using
male mating pheromones to lure females into traps and no studies have described female
behaviors around pheromone-baited traps. This thesis describes in-stream trapping
experiments, which demonstrate that more than 50% of ovulating females are captured in
traps baited with spermiating males and in traps baited with water conditioned by
spermiating males (spermiating male washings), whereas unbaited traps do not capture
females. Additionally, the behavior of females near traps baited with pulsed spermiating
male washings was characterized by more downstream and side-stream movements than
females near traps with continuous washings. Furthermore, results demonstrate that
ovulating females with occluded olfactory organs are unable to locate males in spawning
streams. This thesis conclusively shows that traps baited with spermiating males and
spermiating male washings capture significant numbers of females, that olfaction is used
to detect pheromones, and that females may use pheromone plume structure to locate the
exact source of pheromones. These results support the utility of mating pheromones to

manage sea lamprey in the Great Lakes.

I dedicate this research in loving memory of my older brother Todd Johnson who spurred
my interest in hunting and fishing. Todd was tragically killed while this thesis was being

drafted.

iii

ACKNOWLEDGMENTS

I thank the Great Lakes Fishery Commission for funding and enthusiastic support
of this project. I also thank the staffs of the United States Geological Survey, Hammond
Bay Biological Station and the United States Fish and Wildlife Service, Marquette
Biological Station, for providing sea lampreys, laboratory space, and supplies. I thank
my advisor Dr. Weiming Li and members of my graduate committee, Drs. Thomas Coon,
Chris Goddard, Kay Holekamp, and Michael Sieflces for technical assistance and research
guidance. Additionally, I thank Dolly Trump and Lydia Lorenz for access to their private
property to conduct field experiments. I extend special thanks to my research assistants:
Mandy Karsten, Kile Kucher, Mark Luehring, David Partyka,Jesse Semeyn, and Staci

Zalewski, for their hard work and dedication to the project.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vii
LIST OF FIGUES ................................................................................... ix
CHAPTER 1
INTRODUCTION .................................................................................... 1
Sea lamprey invasion and control in the Great Lakes ................................... 2
Sea lamprey life history ..................................................................... 4
Current understanding of sea lamprey pheromones ..................................... 5
Questions critical to the application of mating pheromones in sea lamprey
management .................................................................................. 7
Questions addressed in this thesis ......................................................... 7
References .................................................................................... 9
CHAPTER 2
MATING PHEROMONE RECEPTION AND INDUCED BEHAVIOR IN
OVULATING FEMALE SEA LAMPREYS ..................... ' .............................. l 2
Abstract ...................................................................................... 13
Introduction ................................................................................. 14
Methods ...................................................................................... 17
Experimental animals ............................................................. 17
Experiment 1. Do traps baited with water conditioned by spermiating
males capture ovulating female sea lampreys? ............................... 17
Test site and equipment ................................................. 18
Experimental design ...................................................... 21
Experiment 2. Is a pulsed pheromone plume as effective as a continuous
pheromone plume? ............................................................... 23
Experiment 3. Are naris-plugged ovulating females attracted to water
conditioned by spermiating males in a two-choice maze? .................. 24
Naris-plugging and control treatment procedures .................... 24
Two-choice maze procedures ........................................... 25
Experiment 4. Can naris-plugged ovulating females locate spermiating
males in a spawning stream? .................................................... 26
Experimental site and equipment ....................................... 26
Experimental design ...................................................... 26
Results ....................................................................................... 29
Discussion .................................................................................... 36
Management implications ........................................................ 38
Acknowledgments .......................................................................... 41
References ................................................................................... 42

APPENDICES

Appendix A. Behavior of naris-plugged ovulating female sea lampreys among

spawning conspecifics .................................................................... 46
Appendix B. Capture of ovulating female sea lampreys in traps baited with
spermiating male sea lampreys .......................................................... 51
Abstract ............................................................................ 52
Introduction ........................................................................ 53
Methods ............................................................................ 55
Experimental animals ................................................... 55
Experimental test site and equipment ................................. 55
Experimental design and procedures .................................. 57
Results .............................................................................. 61
Discussion .......................................................................... 64
Management implications .............................................. 66
Acknowledgments ................................................................ 68
References .......................................................................... 69
Appendix C. Permission to use published materials ................................... 71

vi

Chapter 2

Table 1.

Table 2.

Table 3.

Appendix A

Table 1.

Appendix B

Table 1.

LIST OF TABLES

Number of ovulating female sea lampreys that entered within 5 m (Within 5
m) and were captured (Captured) in traps baited with continuous
spermiating male washings (Constant) and pulsed spermiating male
washings (Pulsed) within 2 h afler release, and the average time to capture
(Time capture) an ovulating female and the average number of downstream
(DS movements) and sidestream (SS movements) movements of ovulating
females within 5 m of traps baited with continuous and pulsed spermiating
male washings. Lower case letters “2”, “y” and “x” indicate significant
differences between continuous and pulsed treatments. Experiments were
conducted on the Ocqueoc River, Presque Isle County, Michigan, USA,
between 18 June and 8 July 2004 ................................................. 30

Number of naris-plugged ovulating female sea lampreys (NPOF) and control
ovulating female sea lampreys (COF) that showed a preference response to
spermiating male sea lamprey washings (SMW) in a two-choice maze.
Statistical significance (P-value) was determined with a two-tailed
Wilcoxon Signed Ranks Test (W—Value) based on the test statistic and the
number of animals tested (n) ....................................................... 33

Number of control ovulating female sea lampreys (COF) and naris-plugged
ovulating female sea lampreys (NPOF) that were released (11) and located
five spermiating male sea lampreys (Males) placed 65 m upstream (65) and
10 m upstream (10) in the Ocqueoc River, Presque Isle County, Michigan,
USA, between 22 July and 10 August, 2004 .................................... 34

The number of control ovulating females and naris-plugged ovulating
females that swam within 1 m of a spermiating male on a spawning nest
(One Meter), interacted with a spermiating male (Interact), and spawned
with a spermiating male (Spawn) in the Ocqueoc River, Presque Isle
County, Michigan, 2005. (n = sample size) ..................................... 48

Results of experiments using traps baited with spermiating male sea
lampreys to attract ovulating females in the Ocqueoc River, Presque Isle
County, Michigan, USA, between June 27 and August 12, 2003. Three

vii

types of traps were used in the experiments: traps containing spermiating
males, traps containing nonsperrniating males, and traps containing no males
(control). Trials involved groups of five females, single females released
during the day, and single females released at night. The following
abbreviations are used: N = is the number of trials for each experiment, S =
the number of females captured in traps with spermiating males, NS = the
number of females captured in traps with nonsperrniating males, NC = the
number of females not captured in traps, and E = the number of females
captured in empty traps .................... 62

viii

Chapter 2

Figure 1.

Figure 2.

Appendix B

Figure 1.

Figure 2.

LIST OF FIGURES

The 65-m section of the Ocqueoc River, Presque Isle County, Michigan,
USA, used for sea lamprey trapping experiments between 18 June and 8
July 2004. The section of river was enclosed with upstream and
downstream block nets (dashed horizontal lines). An island naturally
divides the river into channel one (C1) and channel two (C2). A sea lamprey
trap was placed in each channel of the river approximately 1 m from the
block net and 0.5 m from the shore (T1 and T2). The arrows represent the
flow of water. Females were released from an acclimation cage (A) at the
downstream block net. As females move upstream (dotted line), they must
enter C1 or C2 ........................................................................ 19

Sea lamprey trap dimensions and trap set used to capture ovulating female
sea lampreys on the Ocqueoc River, Presque Isle County, Michigan, USA,
between 18 June and 8 July 2004. Sea lamprey traps were set in 0.3 m of
water (W), 1 m downstream of a block net (BN), and were placed in a hand
constructed depression in the stream bottom. Rocks approximately 5 cm in
diameter were placed around the traps and in front of the traps to imitate a
sea lamprey nest. Water conditioned with spermiating males was pumped in
the traps (SMW) and created a pheromone plume (P) exiting the
downstream funnel of the trap. Ovulating females (OF) downstream of the
trap would follow the pheromone plume to the trap ............................ 20

The 65-m enclosed section of the Ocqueoc River, Presque Isle County,
Michigan used for trapping experiments between 27 June and 12 August
2003. At the upstream block net, an island naturally divides the river into
two channels (C1 and C2). The arrows represent the flow of water coming
from each channel. A sea lamprey trap was placed in each channel of the
river approximately 0.2 m from the block net and 1.0 m from the shore (T1
and T2). Ovulating females were released from an acclimation cage (A) at
the downstream block net and observed until they entered a trap or the end
of the experiment (12 h from the time of release) .............................. 56

The design and dimensions of sea lamprey traps used on the Ocqueoc River,
Presque Isle County, Michigan between 27 June and 12 August 2003. The
dark arrows represent the current flowing through the trap. This created a
pheromone plume (P) originating directly from the downstream funnel of

ix

the trap. A cage (C) with rocky substrate was placed inside the trap to hold
spermiating male sea lampreys .................................................... 58

CHAPTER 1

INTRODUCTION

Sea lamprey invasion and control in the Great Lakes

Sea lamprey (Petromyzon marinas) invaded the upper Laurentian Great Lakes
during the first half of the 20th century and were a primary contributor to catastrophic
ecological and economical damage to the Great Lakes fisheries (Smith and Tibbles
1980). Sea lamprey entered the upper Great Lakes via the Welland Canal, built to allow
the passage of ships around Niagara Falls (Applegate 1950). Prior to sea lamprey
establishment (19405), the upper Great Lakes produced an annual lake trout (Salvelinus
namaycush) harvest of 7,000 tons. However, in the 19505 and 19605, sea lamprey
predation and overfishing resulted in a 95% reduction in lake trout harvest. Lake trout
were not the only species targeted by sea lamprey; high lamprey scarring rates and drastic
reductions in harvest were also observed in lake Whitefish (Coregonus clupeaformz’s),
catostomids (Catostomus spp. and Moxostoma spp.), walleye (Sander vitreus), and
rainbow trout (Oncorhynchus mykiss) (Smith and Tibbles 1980).

The first attempt to control sea lamprey occurred in the 19405 and 19505 when
mechanical and electrical barriers were used to block access to spawning grounds
(Applegate 1950). Barriers constructed at that time were typically expensive to build and
maintain, and lampreys commonly escaped upstream (Applegate 1951; Smith and
Tibbles 1980). Advances in barrier design and construction in the 19705 and 19805
resulted in the use of smaller, less expensive structures that successfully blocked lamprey
migrations (reviewed in Lavis et a1. 2003). Since the 19805, barriers have become a
primary component of integrated sea lamprey management in the Great Lakes.

Currently, more than 60 barriers are used to block access to lamprey spawning habitat,

trap migrating adults, and provide males for the sterile male release program (Lavis et al.
2003).

In the late 19505 and early 19605, the search for a larval lampricide proved
productive when two compounds selectively toxic to lamprey were identified: 3-
trifluoromethyl-4-nitrophenol (TFM) and 5, 2’-dichloro-4‘-nitrosalicylanilide (Bayer 73)
(Applegate et al. 1961; Howell et al. 1964). Application of TFM and Bayer 73 in the
19605 and 19705 significantly reduced lamprey populations. In Lake Superior tributaries,
lamprey spawning runs dropped by 86% after chemical treatments and similar trends
were reported throughout the Great Lakes (Smith and Tibbles 1980; Pearce et a1. 1980).
However, lampricide application only constitutes a single, temporary, and expensive
method of lamprey control (Smith and Tibbles 1980). Growing concern about the social
acceptance of chemical lampricide treatments and the untreatable nature of some streams,
require that additional control techniques be developed (Christie and Goddard 2003).

In response to this concern, the sterile male release technique was developed as
part of integrated sea lamprey management (Hanson and Manion 1980). Since 1997,
approximately 30,000 adult male sea lampreys have been sterilized and released annually
into the St. Mary’s River (connecting Lake Superior and Lake Huron) resulting in a
theoretical reduction in reproduction of 86% when combined with trapping (Twohey et
al. 2003). All available sterilized males are released into the St. Mary’s River because it
is too large to be completely treated with lampricide and, therefore, has been the source
of more than 90% of the parasitic sea lampreys in Lake Huron (Twohey et al. 2003).
Thus, the remaining 430 lamprey-producing tributaries of the Great Lakes can not be

stocked with sterile males and can only be managed with larnpricides and ban'iers.

Additional sea lamprey control techniques would improve integrated sea lamprey

management in the Great Lakes (Christie and Goddard 2003).

Sea lamprey life history

The sea lamprey (Petromyzon marinas) is a jawless anadromous fish native to the
north Atlantic Ocean with a complex life cycle consisting of larval, parasitic, and
spawning phases (Applegate 1950; Hardisty and Potter 1971). Sea lamprey begin their
life as sedentary filter feeding larvae in freshwater streams. Upon reaching a critical
length of approximately 150 mm, larvae metamorphose (develop eyes, teeth, and a sucker
mouth) into the parasitic phase and migrate downstream to an ocean or lake. Parasitic sea
lamprey are efficient ectoparasites of large fishes and extract blood and lymph from their
host. Afler spending 12 to 18 months as parasites, sea lamprey stop feeding and enter the
spawning phase. Spawning-phase sea lamprey migrate up suitable spawning streams in
the spring and early summer. Selection of spawning streams is influenced by migratory
pheromones released by larval lamprey (Polkinghorne 2001). Sea lamprey require gravel
substrate with unidirectional water flow at speeds of 0.5 to 1.5 m/sec for successful
spawning. Spermiating males typically arrive on the spawning grounds before females,
construct several nests, and release pheromones to attract females (Appelgate 1950; Li et
al. 2002). Males are joined by one or more ovulating females and generally spawn for 1
to 3 days. Spent lampreys die shortly after spawning. Fertilized eggs hatch into larvae

and the life cycle repeats itself.

Sexually mature sea lampreys are highly congregated at specific spawning
habitats in streams and, therefore, may be highly vulnerable to control. Spawning
success could be reduced if sea lampreys could be directly removed from spawning
grounds in traps. It has been hypothesized that traps baited with male mating
pheromones might be used to directly remove females from spawning grounds (Li et al.

2003)

Current understanding of sea lamprey pheromones

Sea lamprey rely on a highly developed olfactory organ throughout their life cycle
to find prey (Kleerekoper 1972) and to locate suitable spawning streams (Polkinghome
2001; Teeter 1980) and potential mates (Li et al. 2002; Teeter 1980). Pheromones,
chemical cues that elicit a specific behavioral or physiological response in conspecifics
(Wyatt 2003), coordinate sea lamprey migration and spawning (Li et a1. 2003; Sorensen
and Vrieze 2003). Larval sea lampreys release migratory pheromones that attract adult
sea lampreys to streams with suitable spawning habitat (Polkinghome 2001). F our
components of the migratory pheromone have been identified as petromyoamine
disulfate, petromyzosterol disulfate, petromyzonol sulfate, and allocholic acid (Sorensen
et al. 2005). Reception of migratory pheromones by adult lampreys is a critical
component of sea lamprey life history. For example, it has been shown that anosmic
lampreys are unable to locate spawning streams (Vrieze and Sorensen 2001).

Spermiating male sea lampreys release mating pheromones into the water via the

gills, which are highly attractive to ovulating females (Li et a1. 2002; Siefl<es et al. 2003).

Two components of the mating pheromone have been identified as 3-keto petromyzonol
sulfate and 3-keto allocholic acid (Li et a1. 2002; Yun et al. 2003). Mating pheromones
influence the locomotive responses of ovulating females. For example, it has been
demonstrated that synthesized 3-keto petromyzonal sulfate can attract ovulating females
70 m upstream to the exact point of release (Siefl(es et a1. 2005). This thesis investigates
the function of sea lamprey mating pheromones because they are a useful model to
investigate how a fish orients to pheromone plumes, how pheromonal communication
influences spawning success, and how pheromonal communication may be exploited to
manage sea lamprey in the Great Lakes.

For 25 years it has been hypothesized that sea lamprey mating pheromones might
be of use in an integrated sea lamprey management program (Smith 1980; Teeter 1980;
Li et al. 2003). In its strategic vision statement, the Great Lakes Fishery Commission
states that one alternative control technique needs be developed by 2010, the most
promising of which is pheromone based (Great Lakes Fishery Commission 2005).
Invasive insect species have been successfully controlled by using pheromone-baited
traps to mass-trap individuals (Howse et al. 1998). Similarly, sea lamprey mating
pheromones could be used to mass-trap ovulating females, redistribute ovulating females
into streams with poor spawning habitat, or disrupt pheromone communication among
spawning lampreys, ultimately reducing their reproductive success (Teeter 1980; Li et al.
2003). However, no field studies have tested the efficacy of using mating pheromones to

trap, redistribute, or disrupt reproduction in ovulating females.

Questions critical to the application of mating pheromones in sea lamprey

management

Although it has been clearly demonstrated that mating pheromones strongly
influence locomotive responses of females in a spawning stream (Li et al. 2002; Sieflces
et al. 2005), research should now focus on how to exploit mating pheromones for use in
sea lamprey management. Many questions must be addressed before field application of
mating-pheromone-based techniques in sea lamprey management. The central question
addresses the feasibility of using pheromones to reduce the reproductive potential of sea
lamprey populations. To answer the central question it must be determined whether sea
lamprey mating pheromones can be used to 1) increase the capture rate of females in
traps, 2) attract females to streams not suitable for spawning, or 3) disrupt pheromone

communication in spawning sea lampreys.

Questions addressed in this thesis

This thesis investigates potential applications of the sea lamprey male mating
pheromone in sea lamprey management by addressing questions pertinent to removing
females from spawning grounds, redistributing females to streams with poor spawning
habitat, and disrupting pheromone communication in spawning sea lampreys. Chapter
two, “Mating Pheromone Reception and Induced Behavior in Ovulating Female Sea

Lampreys”, describes experiments that address the following questions:

1) What is the capture rate of females in traps baited with spermiating

male washings?

2) How do females navigate continuous and pulsed pheromone plumes

and can a pulsed plume be used to capture or redistribute females?

3) Is olfaction the only means by which females detect mating pheromones

and locate spermiating males in spawning streams?

Questions 1 and 2 are critical to understanding the ability of mating pheromones
to lure females into traps or to redistribute females. Question 3 is critical to
understanding the mechanism of pheromone reception in females and the importance of
pheromone reception for mate-finding in spawning streams.

Appendix A, “Behavior of Naris-plugged Ovulating Female Sea Lampreys
Among Spawning Conspecifics”, further investigates the importance of pheromone
reception for female mate-finding on sea lamprey spawning grounds. Appendix B,
“Capture of Ovulating Female Sea Lampreys in Traps Baited with Spermiating Male Sea
Lampreys”, describes the first study to demonstrate that spermiating male odors can lure
females into traps and reports the capture rate of females in traps baited with spermiating

males.

References

Applegate, V. c.1950. Natural history of the sea lamprey (Petromyzon marinas) in
Michigan. U. S. Fish. Wildl. Serv. Spec. Sci. Rep. Fish. Serv. No. 55.

Applegate, V.C. and Smith, BR. 1951. Sea lamprey spawning runs in the Great Lakes,
1950. U. S. Fish. Wildl. Serv. Spec. Sci. Rep. Fish. Serv. no. 61.

Applegate, V.C., Howell, J.H., Moffett, J .W., Johnson B.G.H., and Smith, MA. 1961.
Use of 3-trifloro-methyl-4-nitrophenol as a selective sea lamprey larvicide. Great
Lakes Fish. Comm. Tech. Rep. No. 1.

Christie, GO and Goddard CL 2003. Sea lamprey international symposium: advances in
the integrated management of sea lamprey in the Great Lakes. J. Great Lakes Res.
29: 1-14.

Great Lake Fishery Commission. 2001. Strategic vision of the Great Lakes Fishery
Commission for the first decade of the new millennium? Great Lakes Fish.
Comm. Available: www.glfc.org. (September 2005).

Hanson, L.H. and Manion, R]. 1980. Sterility method of pest control and its potential
role in an integrated sea lamprey (Petromyzon marinas) control program. Can. J.
Fish. Aquat. Sci. 37: 2108-2117.

Hardisty, M.W. and Potter, LC. 1971. The general biology of adult lampreys. In: The
Biology of Lampreys, vol. 1, edited by: Hardisty, M.W. and Potter I.C., Academic
Press, New York, pp. 127-206.

Howell, J .H., King Jr., E.L., Smith, A.L., and Hanson, L.H. 1964. Synergism of 5, 2’-
dichloro-4‘-nitrosalicylanilide and 3-trifloro-methyl-4-nitrophenol in a selective
lamprey larvicide. Great Lakes Fish. Comm. Tech. Rep. No 8.

Howse, P.E., Stevens, ID, and Jones, OT. 1998. Insect pheromones and their use in pest
management. Chapman and Hall, New York.

Kleerekoper, H. 1972. The sense organs. In: The Biology of Lampreys, vol. 2, edited by:
Hardisty, M.W. and Potter I.C., Academic Press, New York, pp. 373-404.

Lavis, D.S., Hallett, A., Koon, E.M., and McAuley, TC. 2003. History of and advances
in ban'iers as an alternative method to suppress sea lampreys in the Great Lakes. J.
Great Lakes Res. 29: 362-372.

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

Li, W., Scott, AP, and Siefl(es, M.J. 2003. Sex pheromone communication in the sea
lamprey: implications for integrated management. J. of Great Lakes Res. 29: 85-
94.

Pearce, W.A., Braem, R.A., Dustin, S.M., and Tibbles, J.J. 1980. Sea lamprey
(Petromyzon marinas) in the lower Great Lakes. Can. J. Fish. Aquat. Sci. 37:
1802-1810.

Polkinghorne, C.N., Olson, J.M., Gallaher, D.G., and Sorensen, PW. 2001. Larval sea
lamprey release two unique bile acids to the water at a sufficient rate to produce a
detectable riverine pheromone plumes. Fish. Physiol. Biochem. 24: 15-30.

Siefltes, 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.
Can. J. Fish. Aquat. Sci., 60: 23-31.

Siefkes, M.J., Winterstein, S., and Li, W. 2005. 3-keto petromyzonol sulfate specifically
attracts ovulating female sea lamprey (Petromyzon marinas). Anim. Behav. In
press.

Smith, BR. 1980. Introduction to the proceedings of the 1979 sea lamprey international
symposium (SLIS). Can. J. Fish. Aquat. Sci. 37: 1585-1587.

Smith, BR, and Tibbles, J .J . 1980. Sea lamprey Petromyzon marinas in Lakes Huron,
Michigan, and Superior: history of invasion and control, 1936-78. Can. J. Fish.
Aquat. Sci. 37: 1780-1801.

Sorensen, P.W., Fine, J .M., Dvomikovs, V., Jeffery, C.S., Shao, R, Wang, J ., Vrieze, L.
A., Anderson, K. A., and Hoye, T. 2005. Mixture of new sulfate steroids functions
as a migratory pheromone in the sea lamprey. Nature Chem. Biol. In press.

Sorensen, P.W., and Vrieze, LA. 2003. The chemical ecology and potential application
of the sea lamprey migratory pheromone. J. Great Lakes Res. 29: 66-84.

Teeter, J. 1980. Pheromone communication in sea lampreys Petromyzon marinus:
implications for p0pulation management. Can. J. Fish. Aquat. Sci. 37: 2123-2132.

Twohey, M.B., Heinrich, J .W., Seelye, J .G., Fredricks, K.T., Bergstedt, R.A., Kaye,
C.A., Scholefield, R.J., McDonald, RB, and Christie GO 2003. The sterile-
male-release technique in Great Lakes sea lamprey management. J .Great Lakes
Res. 29: 410-423.

10

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

Wyatt, TD. 2003. Pheromones and Animal Behavior: Communication by Smell and
Taste. Cambridge University Press, New York.

Yun, S-S., Scott, AP, and Li, W. 2003. Pheromones of the male sea lamprey,

Petromyzon marinas: structural studies on a new compound, 3-keto allocholic
acid, and 3-keto petromyzonol sulfate. Steroids 68: 297-304.

11

CHAPTER 2

MATING PHEROMONE RECEPTION AND INDUCED BEHAVIOR IN
OVULATING FEMALE SEA LAMPREYS

Johnson, N. S., Luehring, M. A., Siefl<es, M. J ., and Li, W. 2005. Mating pheromone

reception and induced behavior in ovulating female sea lampreys. North American
Journal of Fisheries Management. in press.

12

ABSTRACT

This study was conducted to determine (how ovulating female sea lampreys
respond to water conditioned with spermiating males (spermiating male washings) and
how trap efficiency can be improved through their use. The capture rate of ovulating
female sea lampreys was observed in traps baited with continuous or pulsed spermiating
male washings. The behavior of ovulating females around baited traps was quantified.
Within 2 h, traps baited with continuous spermiating male washings captured 52% of
ovulating females (n=27) and traps baited with pulsed washings captured 28% (n=25) of
ovulating females. Unbaited traps did not capture ovulating females. The behavior of
females near traps baited with pulsed spermiating male washings was characterized by
significantly more downstream and side-stream movements than females near traps with
continuous washings. We occluded the olfactory organ of ovulating females and tested if
they were attracted to spermiating male washings in a two-choice maze and if they could
locate spermiating males in a spawning stream. Ovulating females with occluded
olfactory organs were unable to locate spermiating males in a spawning stream.
Furthermore, anosmic females were not attracted to spermiating male washings in a two-
choice maze. We conclude that traps baited with spermiating male washings are able to
capture females and that females may use the structure of the pheromone plume to locate
the exact source of pheromones. It is likely that olfaction is the only means for ovulating

females to detect a pheromone that is released by spermiating males.

13

INTRODUCTION

Sea lamprey Petromyzon marinus invaded the upper Laurentian Great Lakes
during the first half of the 20th century and inflicted catastrophic ecological and
economical damage to the Great Lakes fisheries (Smith and Tibbles 1980). The
destruction caused by the sea lamprey prompted one of the most extensive efforts to
control an exotic vertebrate species in North America (Smith and Tibbles 1980).
Currently, sea lamprey populations in the Great Lakes are controlled with lampricide
treatments, sea lamprey barriers, trapping, and sterile-male releases (Christie and
Goddard 2003). The integration of these control techniques has reduced sea lamprey
populations to levels that allow the Great Lakes ecosystem to support productive
salmonid fisheries (Heinrich et al. 2003; Lavis et al. 2003; Morse et al. 2003). Sea
lamprey control remains highly dependent on lampricide treatments that kill filter-feeding
ammocoete larvae in natal streams (Christie et al. 2003). Growing concern about the
social acceptance of chemical lampricide treatments, increasing cost of larnpricides, and
the untreatable nature of some streams requires that new control techniques be developed
and integrated into the Great Lakes sea lamprey management program (Christie and
Goddard 2003). Mating pheromones, commonly integrated into insect control programs,
have been successfully used to monitor, mass-trap, and disrupt reproduction in pest
populations (Howse et al. 1998) and may be useful in lamprey control programs.

Sea lamprey mating pheromones have potential in controlling sea lampreys in the
Great Lakes (Teeter 1980; Li et al. 2002; Li et al. 2003). Spermiating male sea lampreys

release the mating pheromone, 3-keto petromyzonol sulfate, at high rates that attract

l4

ovulating females (Li et al. 2002). Johnson et al. (2005) baited traps with spermiating
males, found that over 70% of ovulating females were captured in baited traps, and
concluded that traps baited with spermiating males may be used to remove females from
spawning grounds. It is not known if females used other sensory modalities to locate
males placed in traps. Furthermore, pheromone-baited traps could be designed more
efficiently (Carde et al. 1998) if pheromone induced behaviors are described, and
schemes to disrupt pheromone communication may become apparent if pheromone
reception in ovulating females is understood (Carde et al. 1990, Sanders 1996).
Currently, the behavior of ovulating females near pheromone-baited traps is poorly
described (Johnson et al. 2005), and the physiological mechanisms of pheromone
reception are not fully identified in ovulating female sea lampreys (Li et al. 2003).

Sea lampreys are believed to detect pheromones through the olfactory organ via a
single dorsal nasopharyngeal opening (Li et al. 1995; Siefl<es and Li 2004). The
olfactory epithelium of adult sea lampreys has been shown to have highly independent
receptor sites for mating pheromones, migratory pheromones and other bile acids (Li and
Sorensen 1997; Siefl<es and Li 2004). Vrieze and Sorensen (2001) showed that migratory
sea lampreys with impaired olfactory systems were not attracted to sea lamprey migratory
pheromones and showed little ability to locate spawning streams. It is not known to what
extent olfaction mediates mating pheromone reception and mate finding in ovulating
female sea lampreys (Li et al. 2003).

The objectives of this study were to 1) determine if ovulating female sea lampreys

could be lured into traps baited with water conditioned with spermiating males, 2)

15

describe behaviors of ovulating females near male pheromone-baited traps, 3) determine

if ovulating females use their olfactory organs to locate spermiating males.

16

METHODS

Experimental animals

Sea lampreys were captured by hand or in mechanical traps fi'om Lake Michigan
and Lake Huron tributaries from May through July 2004. Females were identified by
their sofi abdomen and were separated from males identified by their dorsal ridge
(Vladykov 1949). Furthermore, adults were classified as spermiating males and
ovulating females if milt and eggs, respectively, were expressed by manual pressure
(Siefkes et al. 2003). Spermiating males and ovulating females were used for
experimental purposes and were stored in separate 150 L flow through tanks at
temperatures ranging from 15 to 22 °C. Non-spermiating males and pre-ovulating
females were stored in separate 1000 L flow through tanks at temperatures ranging from
4 to 14 oC. Non-spermiating males and pre-ovulating females were checked weekly for
spermiation and ovulation. To induce female ovulation, several pre-ovulating females
were placed in cages with spermiating males in the Ocqueoc River, a Lake Huron
tributary in Presque Isle County, Michigan, at temperatures ranging from 14 to 25 °C.
Additionally, several pre-ovulating females were stored with spermiating males in a 1000

L flow through tank at 16 °C to induce maturation.

Experiment 1. Do traps baited with water conditioned by spermiating males

capture ovulating female sea lampreys?

17

Test site and equipment

Experiments were conducted above the lamprey barrier on the Ocqueoc River
(Figure 1). Historically, the Ocqueoc River produced significant spawning runs of sea
lampreys (Applegate 1950); however, above the barrier, no sea lampreys have been
observed and the stream contains suitable physical characteristics for spawning. A 65 m
section of the river was enclosed using two block nets. At the upstream block net the
river is divided into two distinct channels with nearly equal discharge of 0.5 m3/sec. The
two channels converge and mix in the middle of the enclosed stream. The stream is a
single channel with an average discharge of 1.01 m3/sec at the downstream barrier.
Stream flow was measured weekly or after significant precipitation events using a Marsh-
McBirney (Marsh-McBimey Incorporated, Fredrick, Maryland) flow meter.

Two identical sea lamprey traps (0.359 m3) were used to capture ovulating
females (Figure 2). A trap was placed in each channel of the stream in approximately 0.3
m of water. Traps were placed 1 m below the upstream barrier and 0.5 m away from the
near shore. The long axes of the traps were positioned parallel to the current to create a
pheromone plume exiting the downstream funnel of the trap. The average velocity of
water flowing through the downstream funnel was 0.24 m/sec. Traps were placed in a
depression in the stream bottom approximately 0.1 m deep and rocks approximately 5 cm
in diameter were placed in front of the trap to imitate a sea lamprey nest. Setting

pheromone-baited traps in hand-constructed spawning nests makes sense biologically

l8

 

Figure 1. The 65-m section of the Ocqueoc River, Presque Isle County, Michigan, USA,
used for sea lamprey trapping experiments between 18 June and 8 July 2004. The section
of river was enclosed with upstream and downstream block nets (dashed horizontal
lines). An island naturally divides the river into channel one (Cl) and channel two (C2).
A sea lamprey trap was placed in each channel of the river approximately 1 m from the
block net and 0.5 m from the shore (T1 and T2). The arrows represent the flow of water.
Females were released from an acclimation cage (A) at the downstream block net. As
females move upstream (dotted line), they must enter C1 or C2.

19

SM BN

 

OF 1)

Van-14mm /' 0.48 m

 

 

 

 

 

Figure 2. Sea lamprey trap dimensions and trap set used to capture ovulating female sea
lampreys on the Ocqueoc River, Presque Isle County, Michigan, USA, between 18 June
and 8 July 2004. Sea lamprey traps were set in 0.3 m of water (W), l m downstream of a
block net (BN), and were placed in a hand constructed depression in the stream bottom.
Rocks approximately 5 cm in diameter were placed around the traps and in front of the
traps to imitate a sea lamprey nest. Water conditioned with spermiating males was
pumped in the traps (SMW) and created a pheromone plume (P) exiting the downstream
funnel of the trap. Ovulating females (OF) downstream of the trap would follow the
pheromone plume to the trap.

20

because spermiating males initiate nest building (Applegate 1950) and release
pheromones that guide females to their nest (Teeter 1980). Additionally, placing traps in
hand-constructed spawning nests lowers the funnel of the trap making it easier for
females to enter as they search along the stream bottom. While we felt that these trap
modifications aided capture efficiency, we did not explicitly test the utility of the
modifications.

Water conditioned by five spermiating males (spermiating male washings) was
used to bait sea lamprey traps. Spermiating male washings were prepared immediately
prior to experimentation by placing five spermiating males in a 25 L bucket of water for
2.5 h. A peristaltic pump was used to apply washings to a trap at a rate of 167 ml/min
(25 L/2.5 h) for 2.5 h. Therefore, the amount of pheromones pumped into a trap over 2.5
h was equal to the amount of pheromones released by five spermiating males in 2.5
hours. More specifically, based on the estimated release rate of 3-keto petromyzonol
sulfate (3kPZS) by a spermiating male, approximately 500 ug'hl‘animal'1 (Y un et al.
2002), and the average streamflow, 1.01 m3/sec, the average in stream concentration of
3kPZS when spermiating male washings were applied to a trap was 1.5 X 10'12 M. This
concentration is very close to the detection threshold of 3kPZS as determined by both
electrophysiological (Siefkes and Li 2004) and behavioral (Sieflces et al. in press) assays.
Ovulating females were released from an acclimation cage (1 m3) placed at the
downstream barrier. The acclimation cage was constructed of 1 cm plastic mesh stapled

to a wood frame.

Experimental design

21

Studies were conducted from 18 June to 8 July 2004, between 0900 hours and
1300 hours in water temperatures ranging from 17 to 20 °C. Ovulating females were
fitted with external radio tags (Advanced Telemetry System, Isanti, Minnesota) according
to Siefl<es et al. (2003) 15 h prior to experimentation. Females were transported to the
Ocqueoc River and placed in the acclimation cage 12 h prior to experimentation.
Spermiating male washings were applied to a randomly chosen (by flipping a coin) trap
and river water was applied to the other trap. Spermiating male washings and river water
were introduced to the traps 30 min prior to female release. After 30 min, the acclimation
cage was opened and ovulating females were allowed to swim out. Five ovulating
females were simultaneously released in all trials but one, in which only two ovulating
females were released due to a shortage of experimental animals. It was assumed that the
behavior of each ovulating female released was independent from other ovulating
females because Johnson et al. (2005) found no significant difference in the capture rate
of individually released ovulating females and simultaneously released females.
Furthermore, Sieflces et al. (in press) found that ovulating females released in groups do
not move in synchrony. Ovulating females were visually observed for 2 h. If females
were not visible, they were tracked with a directional radio antenna and receiver (Lotek
Engineering Incorporated, Newmarket, Ontario, Canada). The 5 m radius of each trap
was marked on the stream bottom with flagging stakes. The time a female entered within
a 5 m radius of the trap and the time of capture were recorded. When a female entered
within 5 m of the baited trap, their behaviors, including downsteam and side-stream
movements, were recorded. A downstream movement was defined as a continuous 2 m

or greater movement downstream with less than 2 m side-stream progress. A side-stream

22

movement was defined as a continuous movement perpendicular to streamflow greater
than 2 m with less than 2 m downstream or upstream progress. The distance a female
traversed downstream and side-stream was visually estimated. The capture rate of

ovulating females in traps baited with spermiating male washings was determined.

Experiment 2. Is a pulsed pheromone plume as effective as a continuous pheromone

plume?

Experiment two was conducted at the same location using the same equipment
and design as experiment one. In this experiment, spermiating male washings were
pulsed into a trap in a pattern of on for 1 min and off for l min. When the odor was
applied to a trap, the in-stream concentration of 3kPZS was approximately 1.5 X 10'12 M.
Only 12.5 L of the 25 L of washings were applied to the trap during an experiment
because the washings were pulsed.

Trials were conducted from 22 June to 7 July 2004, between 0900 hours and 1300
hours in water temperatures ranging from 17 to 20 0C. A z-test for two proportions was
used to compare the proportion of ovulating females that left the acclimation cage,
entered within a 5 m radius of the baited trap, and were captured when washings were
continuously applied to a trap and when they were pulsed into a trap. A two way t-test
assuming equal variance was used to compare the average time of capture for females in
traps baited with continuous washings and pulsed washings. A two way t-test assuming

unequal variance was used to compare the average number of downstream movements

23

and side-stream movements of females that entered within 5 m of traps baited with

continuous washings and pulsed washings.

Experiment 3. Are naris—plugged ovulating females attracted to water conditioned

by spermiating males in a two-choice maze?

N aris-plugging and control treatment procedures

Ovulating female sea lampreys were treated with a naris-plug or a control
treatment 12 h before experimentation. Naris-plugged animals were removed from water
and Stern Vantage Quick Light Body (Sterngold, Boston, Massachusetts), a dental
impression adhesive, was injected into the olfactory cavity to occlude the nasopharyngeal
opening. Next, a drop of Vetbond (Minnesota Mining and Manufacture, St Paul,
Minnesota) was applied in the naris to adhere to the Stern Vantage and completely block
the movement of water through the naris. The Stern Vantage and vetbond were allowed
to air dry for 10 s before females were returned to water. Naris-plugging procedures took
approximately 1 min.

A control treatment was applied to non-naris plugged females to control for
olfactory irritation and the nose-plugging procedure stress. Control ovulating females
were removed from water and a 5 mm tip of a 1-200 uL volume pipet-tip (Dot Scientific
Incorporated, Lippincott Burton, Michigan) was inserted into the nasopharyngeal

opening. Immediately following, vetbond was applied around the naris, but not in the

24

naris. Vetbond was allowed to air dry for 10 s before females were returned to water.

Control treatment procedures took approximately 1 min.

Two-choice maze procedures

Experiments were conducted from 20 July to 29 July 2004, between 0700 hours
and 1300 hours in water temperatures ranging from 16 to 23 °C. Sea lamprey preference
response in a two-choice maze can be used to assess attraction to odors (Li et al. 2002,
Sieflces et al. 2003). Therefore, a two-choice maze was used to assess the attraction of
naris-plugged and control ovulating females to water conditioned by spermiating males.
A two-choice maze was constructed on the bank of the Ocqueoc River and was of exact
dimensions of the maze used by Li et al. (2002) and Siefl<es et al. (2003). River water
was pumped into the maze to create a flow of approximately 0.07 m3/sec. In each test,
the preference behavior of either a naris-plugged ovulating female or a control ovulating
female was recorded before and after the introduction of 10 L of water conditioned with
one spermiating male for 1 h (spermiating male washings). Spermiating male washings
were applied after a 20 min control period to a random arm of the maze at a rate of 400
ml/min for 25 min. Data were collected and analyzed according to the procedure
described in Li et al. (2002) and Siefl<es et al. (2003). Briefly, the time spent (preference)
in the treatment and the control arm of the maze before and after the introduction of
spermiating male washings were summed by a naive observer. A two-tailed Wilcoxon
signed rank test (Rao 1998) was used to determine significant differences in preference

between naris-plugged and control ovulating females.

25

Experiment 4: Can naris-plugged ovulating females locate spermiating males in a

spawning stream?

Experimental site and equipment

Naris-plugged and control ovulating females were released to determine if they
could locate five spermiating males 10 m or 65 m upstream. This experiment was
conducted at the same location as experiments one and two (Figure 1). Ovulating
females were released from an acclimation cage (1 m3). A block net (1 cm plastic mesh)
was placed 5 m downstream of the acclimation cage to prevent females fi'om moving
downstream. No block net was placed upstream of the acclimation cage to obstruct
upstream movement. Spermiating males were held in a cage (0.002 m3) constructed of 1
cm plastic mesh stapled to a wood frame and were placed 10 m or 65 m upstream of the
acclimation cage in a hand constructed depression in the stream bottom 0.1 m deep to

imitate a sea lamprey nest.

Experimental design

Experiments were conducted from 22 July to 10 August 2004, between 0700
hours and 1800 hours in water temperatures ranging from 18 to 24 °C. Naris-plugged and
control ovulating females were fitted with external radio tags (Advanced Telemetry
Systems, Isanti, Minnesota) according to Siefl<es et al. (2003) at least 12 h prior to

experimentation.

26

Ovulating females were held in the acclimation cage prior to experimentation.
Five spermiating males were randomly placed 10 m or 65 m upstream of the ovulating
females 30 min prior to release. Spermiating males placed 10 m upstream of ovulating
females were positioned so that the pheromone plume passed directly through the
acclimation cage. Spermiating males positioned 65 m upstream of ovulating females
were randomly placed in one of two channels of the river by flipping a coin. Naris-
plugged and control ovulating females were released together to control for
environmental variability between tests. Either two naris-plugged and three control
ovulating females were released or three naris-plugged and two control ovulating females
were released during each trial. When the males had been in the stream for 30 min, the
acclimation cage was opened and females were allowed to swim out. Females were
visually observed for 2 h and the location and behavior of each female was recorded. If
females were not visible, they were tracked with a directional radio antenna and receiver
(Lotek Engineering Incorporated, Newmarket, Ontario, Canada). Females that came
within 0.5 m of the spermiating males and searched around the cage for longer than 1 min
were deemed to have located the spermiating males. Females that searched around the
cage for longer than 30 min were removed from the stream. Females that swam more
than 20 m upstream of the spermiating males were removed from the stream.

A z-test for two proportions was used to compare the proportion of naris-plugged
and control ovulating females that moved upstream when males were at 65 m. A Fisher’s
Exact Test was used to compare the proportion of naris-plugged and control ovulating
females that moved upstream when males were at 10 m. A Fisher’s Exact Test was used

to compare the number of naris-plugged and control ovulating females that located five

27

spermiating males at 10 m and 65 m. It was assumed that the behavior of each ovulating
female released was independent from other ovulating females because Sieflces et al. (in

press) found that females released simultaneously do not interact or move in synchrony.

28

RESULTS

Twenty-seven individual ovulating females were released when spermiating male
washings were applied continuously to a trap and 25 individual ovulating females were
released when spermiating male washings were pulsed into a trap. Ovulating females
were captured in traps baited with continuous and pulsed spermiating male washings but
not in traps baited with river water (Table 1). When washings were continuously applied
to a trap, significantly more ovulating females were captured within 2 h (capture rate:
52%) than in traps baited with pulsed washings (capture rate: 28%; z = 2.11, n = 27 and
25 respectively, P = 0.035). Traps baited with continuous washings and pulsed washings
lured approximately equal proportions of ovulating females to within 5 m of the baited
trap (z = 0.739, n = 27 and 25 respectively, P = 0.460; Table 1).

During the 2 h test period, two-thirds of the ovulating females moved upstream
toward the channel with the trap baited with continuous or pulsed spermiating male
washings. One-third of the ovulating females did not move upstream during experiments.
The time of capture ranged from 15 min to 2 h after release. Eight percent of the females
released were still progressing upstream toward the baited trap when the experiment
ended at 2 h. The average time to capture in a trap baited with continuous washings (40.5
min, SD 27.2) was not different from the average time to capture in a trap baited with
pulsed washings (41.7 min, SD 34.9) (Table 1) (t = 0.803; df = 19; P = 0.432). Four of
the 14 females that were captured in traps baited with continuous washings immediately
entered the trap without resting or searching around the trap. Two of the seven females

that were captured in traps baited with pulsed washings immediately

29

Table 1. Number of ovulating female sea lampreys that entered within 5 m (Within 5 m)
and were captured (Captured) in traps baited with continuous spermiating male washings
(Constant) and pulsed spernriating male washings (Pulsed) within 2 h after release, and
the average time to capture (Time capture) an ovulating female and the average number
of downstream (DS movements) and sidestream (SS movements) movements of
ovulating females within 5 m of traps baited with continuous and pulsed spermiating
male washings. Lower case letters “2”, “y” and “x” indicate significant differences
between continuous and pulsed treatments. Experiments were conducted on the Ocqueoc
River, Presque Isle County, Michigan, USA, between 18 June and 8 July 2004.

 

Observation Constant Pulsed
n 27 25
Within 5 m 16 14
Captured z 14 7
Time capture 41 42
DS movements y 0.38 6.08
SS movements x 0.19 1.77

 

30

entered the trap without resting or searching around the trap. However, most ovulating
females spent several minutes searching around the trap before being captured. Common
behaviors near baited traps included resting in front of the trap, rubbing on the sides of
the trap, swimming under the trap, passing in front of the funnel, moving rocks,
downstream movements, and side-stream movements. The average time an ovulating
female spent within 5 m of a trap baited with continuous washings before being captured
was 8.3 min (SD 9.6) and the average time spent within 5 m of a trap with pulsed
washings before being captured was 8.7 min (SD 6.3). Ovulating females always entered
the trap through the downstream funnel and never entered the trap as a result of an

interaction with the upstream barrier.

The behavior of ovulating females near traps was strongly influenced by pulsing
spermiating male washings. First, ovulating females only entered traps baited with
pulsed washings during the periods when the washings were applied to the trap.
Secondly, ovulating females near traps with pulsed washings had significantly more
downstream movements (t = 3.55; df = 12; P = 0.004) and side-stream (t = 2.49; df = 13;
P = 0.027) movements than females near traps with continuous washings (Table l).
Eighty-six percent of females that entered within 5 m of a trap baited with pulsed
washings moved upstream when washings were applied to the trap and moved
downstream or side-stream when the washings were not applied to the trap. The
culmination of successive upstream and downstream movements resulted in cyclic
movement patterns away from the trap and towards the trap. F ifty—four percent of the
females that exhibited cyclic movement patterns were not captured in traps baited with

pulsed washings.

31

Eleven individual naris-plugged ovulating females and 12 individual control
ovulating females were tested in a two-choice maze for attraction to spermiating male
washings. Naris-plugged ovulating females did not show a preference for spermiating
male washings in a two-choice maze (Wilcoxon Signed Ranks Test; n = 11; P > 0.250;
Table 2). Control ovulating females showed a significant preference for spermiating
male washings in a two choice maze (Wilcoxon Signed Ranks Test; n = 12; P < 0.010;
Table 2).

Fourteen individual naris-plugged ovulating females and 14 individual control
ovulating females were released when spermiating males were placed 10 m upstream and
65 m upstream of ovulating females. Naris-plugged ovulating females were unable to
locate five spermiating males 65 m or 10 m upstream of females (Table 3). When males
were placed 65 m upstream, 36% of naris-plugged females swam past the males, 21%
swam upstream but did not make it to the males, and 43% remained in the acclimation
cage or swam to the downstream barrier. When males were placed 10 m upstream, 42%
of naris-plugged females swam past the males and 58% remained in the acclimation cage
or swam to the downstream barrier. Control ovulating females were able to locate five
spermiating males 65 m and 10 m upstream of females. When males were placed 65 m
upstream, 50% of control ovulating females located spermiating males, 14% swam past
the males, 14% swam upstream but did not make it to the males, and 21% remained in
the acclimation cage or swam to the downstream ban'ier. When males were placed 10 m
upstream, 71% of control ovulating females located spermiating males, 21% swam past
the males, and 7% remained in the acclimation cage or swam to the downstream barrier

(Table 3). The proportion of naris-plugged and control females that moved upstream was

32

Table 2. Number of naris-plugged ovulating female sea lampreys (NPOF) and control
ovulating female sea lampreys (COF) that showed a preference response to spermiating
male sea lamprey washings (SMW) in a two-choice maze. Statistical significance (P-
value) was determined with a two-tailed Wilcoxon Signed Ranks Test (W—Value) based
on the test statistic and the number of animals tested (n).

 

Test Subject n SMW W-value P-value
NPOF 1 1 3 27 NS
COF 12 10 73 <0.01O

 

33

Table 3. Number of control ovulating female sea lampreys (COF) and naris-plugged
ovulating female sea lampreys (NPOF) that were released (n) and located five
spermiating male sea lampreys (Males) placed 65 m upstream (65) and 10 m upstream
(10) in the Ocqueoc River, Presque Isle County, Michigan, USA, between 22 July and 10
August, 2004.

 

Animal Distance n Males
COF 65 14 7

NPOF 65 14 O
COF 10 14 10

NPOF 10 14 O

 

34

not significantly different when males were placed at 65 m (z-test, P = 0.106). The
proportion of control females that moved upstream was significantly greater than the
proportion of naris-plugged females that moved upstream when males were placed at 10
m (Fisher’s Exact Test, P = 0.016). Control ovulating females located five spermiating
males significantly more often than naris-plugged ovulating females when males were

placed 10 m and 65 m upstream (Fisher’s Exact Test, P=0.003; P < 0.001).

35

DISCUSSION

Our results demonstrate that spermiating male washings are able to lure ovulating
female sea lampreys into traps. At our experimental site, traps baited with continuous
spermiating male washings captured 52% of ovulating females within 2 h. The capture
rate of females in traps baited with continuous spermiating male washings is similar to
the capture rate of females in traps baited with spermiating males, where 40% of
ovulating females were captured within 30 min and 70% of ovulating females were
captured within 12 h (Johnson et al. 2005). Therefore, our study suggests that
pheromones are a powerful trap bait which could be used instead of baiting traps with
spermiating males. Future studies should investigate differences in capture rates in traps
baited with spermiating males, spermiating male washings, extracted pheromones, and
synthetic pheromones.

We hypothesize that the detection of male pheromones by ovulating females
motivate their upstream movement. Pheromone plumes are described as turbulent,
unpredictable filaments, which become widely spaced as they are carried away fi'om the
source (Keller et al. 2001; Sherman and Moore 2001; Wyatt 2003). In many insect
species, fluctuating pheromone plumes are required for sustained upwind flight (Carde
and Elkinton 1984; Baker and Haynes 1989). In the aquatic environment, crayfish
Orconectes virilis have been shown to approach an odor source more quickly when the
odor plume is turbulent (Moore and Grills 1999; Keller et al. 2001). Similarly, in our
experiment, a pulsed pheromone plume lured ovulating females upstream, and equal

proportions of females 65 m downstream of continuous and pulsed pheromones were

36

lured to within 5 m of the baited trap. This may have occurred because the pheromone
plume of both continuous and pulsed pheromone sources may have only consisted of
random filaments of pheromones 65 m downstream from the source. It is possible, but
has not yet been unequivocally demonstrated, that random widely-spaced filaments of
pheromones may trigger upstream movement in ovulating females.

It is likely that ovulating females rely on pheromone plume structure to locate the
exact source of pheromones. Near a pheromone source, the plume is described as a
continuous burst of pheromones (Baker and Haynes 1989; Zimmer-Faust et a1. 1995;
Keller et a1. 2001 ). In many insect species, continuous burst of pheromones cause the
arrestrnent of upwind progress (Carde and Elkinton 1984; Baker and Haynes 1989).
Similarly, in our experiment, ovulating females typically spent several minutes below the
baited trap before entering and never swam past a pheromone-baited trap. It is possible
that ovulating females slowed upstream movement near baited traps because they
encountered continuous bursts of pheromones indicating they were near the source.
Further evidence to support this hypothesis is that when washings were pulsed into a trap,
ovulating females moved downstream and side-stream when the odor was not applied.
Down and side-stream movement may have occurred because when washings were
suddenly discontinued, the instinctual interpretation was that the odor source moved
downstream or side-stream, but not upstream, because the pheromone bursts did not
become less frequent, but instead stopped completely. Therefore, the female may have
drifted downstream and moved side-stream in an attempt to reencounter the pheromone

plume.

37

Two-choice maze results demonstrate that ovulating females incapable of
olfaction are not attracted to mating pheromones. Our results are consistent with Siefkes
and Li (2004) who hypothesized that olfaction is the primary means of pheromone
detection and characterized pheromone receptor sites in the olfactory epithelium of
female sea lampreys. Our results also parallel with those of Vrieze and Sorensen (2001)
who showed that migratory sea lampreys with occluded olfactory systems were not
attracted to larval sea lamprey washings in a two-choice maze, and showed little ability to
locate spawning streams.

In-stream olfactory occlusion experiments demonstrate that pheromone reception
in sexually mature sea lamprey is essential for locating mates. Locating mates without
pheromonal communication would likely be inefficient because sexually mature sea
lampreys have poor vision (Manion and Hanson 1980) and electroreception is limited to a
few centimeters (Bodznick and Nortcutt 1981). It is also unlikely that males actively
search for females, since males arrive at the spawning grounds before females, initiate
nest building, and actively signal females with pheromones (Applegate 1950, Li et al.
2002; Li et al. 2003). Some insect control programs have exploited the dependency on
pheromonal communication for mate finding by using high concentrations of synthetic
pheromones to disrupt orientation to natural pheromone sources (Carde et al. 1998).
Insect control programs have also developed pheromone antagonists that completely
block pheromone reception and stop pheromone induced behavior (Millar and Rice 1996;

Evenden et al. 1999).

Management implications

38

Pheromone-baited traps are able to capture ovulating females, even when
spermiating males are not placed inside. In this study, more than 50% of ovulating
females were captured within 2 h in traps baited with spermiating male washings. The
capture rate of females in traps baited with spermiating male washings is similar to the
capture rate in traps baited with spermiating males (Johnson et al. 2005). Additionally,
females captured in this experiment never interacted with the upstream barrier.
Therefore, pheromone baited traps may be used to remove ripe females from spawning
grounds without the use of a barrier. Mating pheromones may be applied to traps in three
different manners. First, spermiating male washings could be directly pumped into traps.
For example, excess water from a flow-through tank stocked with spermiating males
could be pumped into a trap at relatively low cost. Secondly, mating pheromones could
be extracted from spermiating male washings and metered into traps. Lastly, synthetic
pheromones, if developed, may be pumped into traps (Li et al. 2003). Future research
should focus on which pheromone application method is most cost effective.

Pulsed mating pheromones may be used to redistribute ovulating female sea
lampreys into tributaries not suitable for spawning. Our results showed that pulsed
washings applied at a rate of on for 1 min off for 1 min, lured equal numbers of females
to within 5 m of the pheromone source. Therefore, if management goals are to
redistribute ovulating female sea lampreys with synthetic mating pheromones into
tributaries or areas not suitable for spawning (Li et al. 2003; Twohey et al. 2003), a
pulsed source may be equally as effective and cost half as much as a continuous

pheromone source.

39

A mating pheromone antagonist, if developed, may reduce the reproductive
success of sea lamprey populations by inhibiting pheromone reception in ovulating
females. In our study, females without the ability to use olfactory pheromone receptor
sites did not exhibit pheromone-induced behavior and were unable to locate spermiating

males in a spawning stream.

40

ACKNOWLEDGEMENTS

The staffs of United States Geological Survey Hammond Bay Biological Station
and United States Fish and Wildlife Service Marquette Biological Station provided
research facilities, sea lampreys, and equipment. Special thanks to Dolly Trump and
Lydia Lorenz who provided access to their land and Mandy Karsten, David Partyka, and
Staci Zalewski who helped conduct this study! The Great Lakes Fisheries Commission

funded this research.

41

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Applegate, V. C. 1950. Natural history of the sea lamprey in Michigan. U. S. Department
of Interior Fish & Wildlife Service, Special Scientific Report Fisheries. No. 55,
Washington, D. C.

Baker, T. C., and K. F. Haynes. 1989. Field and laboratory electroantennographic
measurements of pheromone plume structure correlated with oriental fruit moth
behavior. Physiological Entomology 14:1-12.

Bodznick, D., and R. G. Northcutt. 1981. Electroreception in lampreys: evidence that the
earliest vertebrates were electroreceptive. Science 212:465-467.

Carde, R. T., and J. S. Elkinton. 1984. Field trapping and attractants: methods and
interpretation. Pages 111-129 in H. E. Hummel and T. A. Miller, editors.
Techniques in pheromone research. Springer-Verlag New York Incorporated.
New York.

Carde, R. T. 1990. Principles of mating disruption. Pages 73-92 in R. L. Ridgeway, R. M.
Silverstein, and M. N. Inscoe, editors. Behavior modifying chemicals for insect
management. Marcel Dekker Incorporated, New York.

Carde, R. T., R. T. Staten, and A. Mafra-Neto. 1998. Behaviour of pink bollworrn males
near high-dose, point sources of pheromone in field wind tunnels: insights into
mechanisms of mating disruption. Entomologia Experimentalis et Applicata
89:35-46.

Christie, G. C., and C. I. Goddard. 2003a. Sea lamprey international symposium:
advances in the integrated management of sea lamprey in the Great Lakes. Journal
of Great Lakes Research 29:1-14.

Christie, G. C., J. V. Adams, T. B. Steeves, J. W. Slade, D. W. Cuddy, M. F. Fodale, R. J.
Young, M. Kuc, and M. L. Jones. 2003b. Selecting Great Lakes streams for

lampricide treatment based on larval sea lamprey surveys. Journal of Great Lakes
Research 29:152-160.

Evenden, M. L., G. J. Judd, and J. H. Borden. 1999. Simultaneous disruption of
pheromone communication in Charistoneura rosaceana and Pandemis limitata
with pheromone and antagonist blends. Journal of Chemical Ecology 25:501-517.

Heinrich, J. W., K. M. Mullett, M. J. Hansen, J. V. Adams, G. T. Klar, D. A. Johnson, G.
C. Christie, and R. J. Young. 2003. Sea lamprey abundance and control in Lake
Superior 1957-1999. Journal of Great Lakes Research 29:566-583.

42

Howse, P. E., I. D. Stevens, and O. T. Jones. 1998. Insect pheromones and their use in
pest management. Chapman and Hall, New York.

Keller, T. A., A. M. Tamba, and P. A. Moore. 2001. Orientation in complex chemical
landscapes: spatial arrangement of chemical sources influences crayfish food-
finding efficiency in artificial streams. Limnology and Oceanography 46:238-247.

Lavis, D. S., M. P. Henson, D. A. Johnson, E. M. Koon, and D. J. Ollila. 2003. A case
history of sea lamprey control in Lake Michigan 1979-1999. Journal of Great
Lakes Research 29:584-598.

Li, W., P. W. Sorensen, and D. D. Gallaher. 1995. The olfactory system of migratory
adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to

unique bile acids released by conspecific larvae. Journal of General Physiology
105:569-587.

Li, W., and P. W. Sorensen. 1997. Highly independent olfactory receptor sites for
naturally occurring bile acids in the sea lamprey, Petromyzon marinus. Journal of
Comparative Physiology 180:429-438.

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

Li, W., A. P. Scott, and M. J. Sieflces. 2003. Sex pheromone communication in the sea
lamprey: implications for integrated management. Journal of Great Lakes
Research 29:85-94.

Johnson, N. S., M. J. Sieflces, and W. Li. 2005. Capture of ovulating female sea lampreys
in traps baited with spermiating male sea lampreys. North American Journal of
Fisheries Management 25: 67-72.

Manion, P. J ., and L. H. Hanson. 1980. Spawning behavior and fecundity of lampreys
from the upper three Great Lakes. Canadian Journal of Fisheries and Aquatic
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Millar, J. G., and R. E. Rice. 1996. 5-Decyn-1-y1 Acetate: powerful antagonist of peach
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Moore, P. A., and J. L. Grills. 1999. Chemical orientation to food by the crayfish
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Morse, T. L., M. P. Ebener, E. M. Koon, S. B. Morkert, D. A. Johnson, D. W. Cuddy, J.
W. Weisser, K. M. Mullet, and J. H. Genovese. 2003. A case history of sea
lamprey control in Lake Huron: 1979-1999. Journal of Great Lakes Research
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Rao, P. V. 1998. Statistical research models in the life sciences. Brooks/Cole Publishing
Company, Pacific Grove, California.

Sanders, C. J. 1996. Mechanisms of mating disruption in moths. Pages 333-346 in R. T.
Carde and A. K. Minks, editors. Insect pheromone research. New directions.
Chapman & Hall, New York.

Sherman M. L., and P. A. Moore. 2001. Chemical orientation of brown bullheads,
Ameiurus nebulosus, under different flow conditions. Journal of Chemical
Ecology 27:2301-2318.

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

Sieflres, M. J ., and W. Li. 2004. Electrophysiological evidence for detection and
discrimination of pheromonal bile acids by the olfactory epithelium of female sea
lampreys (Petromyzon marinas). Journal of Comparative Physiology 190:193-
199.

Sieflces, M. J., S. Winterstein, and W. Li. 2005. 3-keto petromyzonol sulfate specifically
attracts ovulating female sea lamprey (Petromyzon marinas). Animal Behaviour.

Smith, B. R., and J. J. Tibbles. 1980. Sea lamprey Petromyzon marinas 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.

Twohey, M. B., P. W. Sorensen, and W. Li. 2003. Possible applications of pheromones in
an integrated program of sea lamprey management. Journal of Great Lakes
Research 29:794-800.

Vladykov, V. D. 1949. Quebec lamprey. 1.-List of species and their economic
importance. Province of Quebec Department of Fisheries, Contribution 26,
Quebec.

Vrieze, L. A., and P. W. Sorensen. 2001. Laboratory assessment of the role of a larval
pheromone and natural stream odor in spawning stream localization by migratory

sea lamprey (Petromyzon marinus). Canadian Journal of Fisheries and Aquatic
Sciences 58:2374-2385.

Wyatt, T. D. 2003. Pheromones and animal behavior: communication by smell and taste.
Cambridge University Press, New York.

Yun, S-S., M. J. Sieflres, A. P. Scott, and W. Li. 2002. Development and application of
an ELSIA for a sex pheromone released by male sea lampreys (Petromyzon
marinas L.). General and Comparative Endocrinology 129:163-170.

Zimmer-Faust, R. K., C. M. Fenelli, N. D. Pentcheff, and D. S. Wethey. 1995. Odor

plums and animal navigation in turbulent water flow: a field study. Biological
Bulletin 188:111-116.

45

APPENDIX A

BEHAVIOR OF NARIS-PLUGGED OVULATIN G FEMALE SEA LAMPREYS
AMONG SPAWNING CONSPECIFICS.

46

Data described in chapter 2 demonstrated that naris-plugged ovulating females are
not attracted to spermiating male washings and do not interact with caged spermiating
males in a spawning stream. However, it was not determined whether naris-plugged
females released among spawning sea lampreys would interact and spawn with
spermiating males. Here I describe an experiment in which 12 naris-plugged females and
21 control females were released directly below a lamprey spawning area in the Ocqueoc
River, Presque Isle County, Michigan. Naris-plugging and control treatment procedures
were conducted as described in chapter 2. From June 6th to June 11th, 2005, in a 115 m
spawning ground, the average number of spermiating males observed on nests during
experimentation was 17 (SE = 6) and the average number of females observed on nests
was 22 (SE = 11). Three naris-plugged females were released each day from June 8th to
June 11‘“, 2005. Five control females were released each day from June 6th to June 8th,
2005, and 6 control females were released on June 9th, 2005. Females were observed for
2 hours after release and the percentage of females that swam within l m of a spermiating
male on a spawning nest, interacted (as defined in chapter 2) with a spermiating male,
and spawned with a spermiating male were recorded.

The percentage of females that swam within 1 m of a spermiating male did not
differ significantly between control and naris-plugged females (Fisher’s Exact Test; p =
0.095). However, naris-plugged ovulating females never interacted or spawned with
spermiating males, while 67 % of control females interacted with a male(s) and 57 % of
control females were observed spawning. Naris-plugged females interacted with
significantly fewer males and spawned significantly less than control females (Fisher’s

Exact Test; p < 0.001; Table 1).

47

Table 1. The number of control ovulating females and naris-plugged ovulating females
that swam within 1 m of a spermiating male on a spawning nest (One Meter), interacted
with a spermiating male (Interact), and spawned with a spermiating male (Spawn) in the
Ocqueoc River, Presque Isle County, Michigan, 2005. (n = sample size)

 

Test Female n One Meter Interact Spawn
Control 21 15 14 12
Naris-plgqged 12 5 0 O

 

48

Results show that mating pheromone reception is critical for mate finding and
reproductive success in female sea lampreys. Chapter 2 demonstrated that ovulating
females incapable of olfaction were not attracted to spermiating male washings and were
unable to locate caged males in a spawning stream. This study shows that naris-plugged
ovulating females do not interact or spawn with free-ranging spermiating males in a
spawning stream and furthermore, spermiating males do not search out naris-plugged
ovulating females to reproduce. These results support the hypothesis that females
actively search for spermiating males by navigating pheromone plumes originating from
spermiating males (Li et al. 2002) and indicate that disrupting pheromonal
communication with a pheromone antagonist or supemorrnal concentrations of
pheromones may reduce the reproductive success of spawning lampreys. (Twohey et al.

2003)

49

REFERENCES

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

Twohey, M.B., Sorensen, P.W., and Li, W. 2003. Possible applications of pheromones in
an integrated sea lamprey management program. J. Great Lakes Res. 29: 794-800.

50

APPENDIX B

CAPTURE OF OVULATING FEMALE SEA LAMPREYS IN TRAPS BAITED
WITH SPERMIATING MALE SEA LAMPREYS

Johnson, N.S., Sieflres, M.J., Li W. 2005. Capture of ovulating female sea lampreys in

traps baited with spermiating male sea lampreys. North Ameficam Journal of Fisheries
Management 25: 67-72.

51

ABSTRACT

This study was conducted as an initial step in the development of a trapping
technique for sexually mature female sea lampreys Petromyzon marinas. Recent research
has demonstrated that spermiating male sea lampreys release a sex pheromone that
attracts ovulating females. This discovery prompted us to hypothesize that traps baited
with spermiating males would capture more ovulating females than empty traps or traps
baited with non-spermiating males. We found that traps baited with spermiating males
captured nearly 74% of the ovulating females released, whereas empty traps and traps
baited with non-spermiating males did not capture any ovulating females. We conclude
that pheromone-baited traps may compliment current sea lamprey management through

direct removal of ripe females from spawning grounds.

52

INTRODUCTION

The sea lamprey Petromyzon marinas invaded the Laurentian Great Lakes and
caused the collapse of numerous economically valuable fish populations (Smith and
Tibbles 1980). Integrated management of sea lamprey is essential to maintain and restore
the Great Lakes ecosystem (Great Lakes Fisheries Commission 2003). Lampricides,
barriers, trapping, and sterile-male releases are used to control sea lamprey populations
(Klar and Young 2002), yet sea lampreys continue to be a significant source of fish
mortality in the Great Lakes (Bergstedt and Scheider 1988; Kitchell 1990). Further, these
techniques can be costly and some have uncertain environmental consequences (Larnsa et
al. 1980; Smith and Tibbles 1980). Additional control techniques will improve sea
lamprey management in the Great Lakes (Hanson and Manion 1980; Smith and Tibbles

1980).

Trapping currently targets sexually immature sea lampreys as they migrate
upstream to their spawning grounds (Great Lakes Fisheries Commission 2003). Captured
females are killed and captured males are used in the sterile-male release program.
Trapping reduces the population of migratory sea lampreys in rivers by an average of
39% in the whole Great Lakes basin and up to 60-80% in some rivers (Klar and Young
2002). We reasoned that if a trapping technique could be developed to firrther reduce the
abundance of ovulating females, the reproductive potential of sea lamprey populations
would be further reduced. This may be logistically challenging because mature sea
lampreys do not appear to move great distances within a stream when compared to

immature sea lampreys en route to spawning grounds (personal observation).

53

Nevertheless, recent advances in our understanding of sea lamprey sex pheromone
communication indicated that baited trapping may provide a solution to this technical

difficulty (Li et al. 2002; Siefkes et al. 2003).

Pheromones play an important role in mate searching and courtship behavior in
sea lamprey (Teeter 1980). Recent research indicates that spermiating males release at
least one sex pheromone that induces a strong preference and searching behavior in
ovulating females (Li et al. 2002; Sieflres et al. 2003). Insect traps baited with specific
female pheromones have successfully captured sexually mature males of the same
species (Beroza and Knipling 1972; Oehlschlager et al. 2003). In principle, lamprey traps
baited with a male pheromone or spermiating males could also be used to capture
ovulating female sea lampreys to reduce the reproductive potential of sea lamprey
populations (Teeter 1980, Li et al. 2002). Our objectives were to determine whether
baiting traps with spermiating males could significantly increase the capture rate of

ovulating females and if trapping rates varied significantly between day and night.

54

METHODS

Experimental animals

Sea lampreys were captured by hand or in traps from Lake Michigan and Huron
tributaries from May until July 2003. Males and females were identified and separated
according to the protocol established by Vladykov (1949). Each sex was further assigned
to one of two maturity classes according to the protocol established by Sieflres et al.
(2003). Males were classified as nonspenniating or spermiating. Females were classified
as preovulating or ovulating. Spermiating males and ovulating females were held in 150-
L flow-through tanks at ambient temperatures ranging from 7°C to 20°C for immediate
experimentation. Nonsperrniating males and nonovulating females were held in 1,000-L,
flow-through tanks at temperatures ranging from 4-8 °C for future experimentation.
Several nonsperrniating males and nonovulating females were held together with
spermiating males and ovulating females in an artificial spawning stream to induce

maturation.

Experimental test site and equipment

Trapping experiments were conducted above the lamprey barrier in a 65-m
section of the Ocqueoc River (a Lake Huron tributary, Presque Isle County, Michigan;

Figure l). The Ocqueoc River is historically known for its large population of spawning

55

 

Figure 1. The 65-m enclosed section of the Ocqueoc River, Presque Isle County,
Michigan used for trapping experiments between 27 June and 12 August 2003. At the
upstream block net, an island naturally divides the river into two channels (C1 and C2).
The arrows represent the flow of water coming from each channel. A sea lamprey trap
was placed in each channel of the river approximately 0.2 m from the block net and 1.0 m
from the shore (T1 and T2). Ovulating females were released from an acclimation cage
(A) at the downstream block net and observed until they entered a trap or the end of the
experiment (12 h from the time of release).

56

sea lampreys (Applegate 1950; Heinrich et al. 1980; Coble et a1. 1990; Houston and
Kelso 1991). However, above the barrier, no sea lampreys were known to be present
(personal observation) and the stream contains suitable physical qualities and habitat for
spawning (Applegate 1950). The 65-m section was enclosed using two block nets. At the
upstream block net, an island naturally divides the river into two distinct channels with
nearly equal discharge (1.6 m3/s). The two channels converge and mix in the middle of
the enclosed river section. Sea lampreys swimming upstream during an experiment must
choose which channel to enter.

Two identical traps (0.359 m3) were used to capture ovulating females in the
enclosed section of the river (Figure 2). A trap was placed in each channel of the river
along the upstream barrier. Each trap was approximately 1 m from the nearest shore and
0.2 m from the upstream block net. The traps were set parallel to the current to create a
pheromone plume originating from the downstream firnnel of the trap (Figure 2). Water
flow through each trap was approximately 0.30 m3/s. Ovulating females were held in an
acclimation cage along the downstream barrier (Figure 1). Males were held in cages
placed inside the trap (Figure 2) to prevent males from escaping or physically interacting

with ovulating females.

Experimental design and procedures

The study was conducted between 27 June and 12 August, 2003. Ovulating
females were fitted with external radio tags (Advanced Telemetry System, Isanti,

Minnesota) according to Siefkes et al. (2003) 24 h prior to experimentation. Five

57

 

0.61

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2. The design and dimensions of sea lamprey traps used on the Ocqueoc River,
Presque Isle County, Michigan between 27 June and 12 August 2003. The dark arrows
represent the current flowing through the trap. This created a pheromone plume (P)
originating directly from the downstream funnel of the trap. A cage (C) with rocky
substrate was placed inside the trap to hold spermiating male sea lampreys.

58

spermiating and five non-spermiating males were randomly assigned to a trap by flipping
a coin. Males in traps and ovulating females were allowed to acclimate in the river for a
minimum of 30 min before experimentation.

The study consisted of two experiments. In the first experiment, a single female
was released in each trial to estimate the trapping rate of individual females. In the
second experiment, five females were released in each trial to estimate the trapping rate
of a group of females. Each experiment further consisted of treatment and control trials.
Treatment trials were conducted by randomly placing five spermiating males in one trap
and five nonsperrniating males in the other. Control trials were conducted with no males
in either trap and estimated the trapping rate of ovulating females using current trapping
techniques. Treatment and control trials were conducted both day and night to
investigate changes in trapping rates under specific lighting conditions (Teeter 1980).
Day trials started at 0900 hours and ended at 2100 hours with ambient water temperatures
of 15.2-29.2°C. Night trials and multiple female releases started at 2100 hours and ended
at 0900 hours with ambient water temperatures of 14.3-29.3°C.

Thirty-nine experimental trials (16 during the day and 23 during the night) and
twenty-four control trials (11 during day and 13 during night) were conducted when
individual females were released. Four treatment trials and one control trial was
conducted during the night when five females were simultaneously released. In all trials,
females were released at the downstream barrier and visually observed. If females were
not visible, they were tracked with a directional radio antenna and receiver (Advanced

Telemetry Systems, Isanti, Minnesota).

59

Ovulating females were allowed 12 h to enter a trap. Females that entered a trap
before 12 h were removed and the experiment was terminated. If a female escaped from
the enclosed river section the trial was counted because the female failed to respond to
the pheromone cue. If a female died, the trial was not counted because we believed the
female was physically unable to respond to the pheromone cue. Ovulating females were
either captured in the trap containing spermiating males, captured in the trap containing
nonspenniating males, captured in the empty trap, or not trapped. Finally, the behaviors
of ovulating females around the traps were observed and described, but not quantified.

Capture rates of ovulating females were calculated by dividing the total number of
females released by the number captured in each trap. A Fisher’s Exact Test was used to
compare night and day trials, and to compare single female releases and five female
releases. If no significant differences were observed between day and night trials and

single- and five- female release trials, the data were combined.

60

RESULTS

Six ovulating females died during treatment trials and one female died during
control trials. These trials were not included in our data analyses. Eight ovulating
females escaped the enclosed river section during treatment trials and three escaped
during control trials. These trials were included in our data analyses.

Trapping rates did not differ significantly between day and night trials (Fisher’s
Exact Test, P = 1.00) or between single- and five-female releases (Fisher’s Exact Test, P
= 0.86; Table 1). Ovulating females were captured in traps containing spermiating males
but not in those with nonspenniating males. During the 53 countable treatment trials,
traps with spermiating males caught nearly 74% of ovulating females released, traps with
nonsperrniating males did not catch any ovulating females released, and approximately
26% of ovulating females were not captured in either trap (Table 1).

Furthermore, ovulating females were quickly captured in the spermiating male
trap. Twenty-one of the 39 females captured entered the trap within 30 min after their
release. The mean time a female was observed entering the trap was 27 min (range, 7-75
min). Observations of 17 of the 39 females captured revealed that 9 swam around the
trap (swimming up and down one or both sides of the trap often touching the trap) for up
to several minutes before entering the trap; where as the other eight swam directly into

the trap.

During the 27 countable control trials, none of the ovulating females were
captured in empty sea lamprey traps (Table 1). Additionally, most ovulating females did

not move upstream when the traps were empty. Of 25 countable control trials, 19

61

Table 1. Results of experiments using traps baited with spermiating male sea lampreys
to attract ovulating females in the Ocqueoc River, Presque Isle County, Michigan, USA,
between June 27 and August 12, 2003. Three types of traps were used in the
experiments: traps containing spermiating males, traps containing nonsperrniating males,
and traps containing no males (control). Trials involved groups of five females, single
females released during the day, and single females released at night. The following
abbreviations are used: N = is the number of trials for each experiment, S = the number
of females captured in traps with spermiating males, NS = the number of females
captured in traps with nonsperrniating males, NC = the number of females not captured in
traps, and E = the number of females captured in empty traps.

 

 

Experimental Control
Trial N S NS NC N E NC
Five releases 19 14 0 5 4 0 4
Single day 16 13 0 3 12 0 12
Single night 18 12 0 6 11 0 11
Total 53 39 O 14 27 0 27

 

62

females did not move upstream, 3 swam halfway up the enclosed river section, and 3

swam to the upstream block net.

63

DISCUSSION

Our results imply that traps baited with spermiating male sea lampreys may be
used to remove ovulating female sea lampreys from spawning grounds. In our
experimental site, the capture rate of ovulating females was increased from zero to more
than 70% by baiting the traps with spermiating males. These results are consistent with
previous studies conducted in mazes and in the field. In a two-choice maze, male sex
pheromones have been shown to induce a strong preference and searching response in
ovulating females (Li et al. 2002; Siefl<es et al. 2003). In field conditions, spermiating
males have been shown to attract ovulating females upstream (Li et al. 2002; Sieflres et
al. 2003); therefore, it is likely that sex pheromones released by spermiating males
attracted ovulating females into the traps. Our study also confirms the “infallible”
practice of French fishermen who used male lampreys to bait traps to capture females
(Fontaine 1938).

An interesting observation in this study was that some females swam around the
spermiating male trap before entering. This behavior may be due to some of the
pheromone plume exiting the side of the trap rather than the funnel. Therefore, females
may have followed the plume to the sides of the trap before finding the funnel and
entering the trap. This pheromone-induced behavior could also be an important
mechanism mediating the attraction of females to males and important to consider when
designing a trapping strategy. More research is needed to find the exact cause of this
behavior and to firrther quantify the behavior of ovulating females around pheromone-

baited traps.

64

Our experiments were conducted under ideal conditions; large-scale studies in
actual spawning situations are needed. First, male sex pheromones have been shown to
attract ovulating females from up to 65 m away (Li et al. 2002; Siefkes et al. 2003; and
data from this study). However, the maximum or effective distance ovulating females are
attracted to pheromone-baited traps has yet to be determined. Second, resident
spermiating males in the field may reduce the capture rate of ovulating females in
pheromone-baited traps. Under the experimental conditions of this study, when no other
sperrrriating males were present in the stream, traps with spermiating males yielded high
capture rates of ovulating females. However, in actual spawning situations, background
pheromones released by conspecific spermiating males may reduce the effectiveness of
pheromone trapping.

Sex pheromone trapping techniques may be improved by additional research.
First, specially designed traps may increase the capture rate of ovulating females (Teeter
1980). Traps designed to deflect the pheromone plume of spermiating males through the
funnel(s) may result in higher capture rates. Second, if synthetic pheromones can be
created that are as potent as spermiating males, they would eliminate the difficulties of
using live spermiating males to bait traps and allow more males to be used in the sterile-
male program. Currently, components of the pheromone mixture, 3-keto petromyzonol
sulfate and 3-keto allocholic acid, are being chemically synthesized and tested in a two-
choice maze to determine if they elicit a searching response similar to that of water

conditioned with spermiating males (unpublished data).

65

Management implications

Sex pheromones have appealing qualities that may benefit sea lamprey
management in the Great Lakes. Although the behavior of ovulating females appears to
be difficult to exploit using current trapping practice (personal observation), our results
suggest that with the use of spermiating males, ovulating females might be efficiently
trapped. In our study, the capture rate of ovulating females was high, pheromones were
naturally released from spermiating males with no apparent environmental damage, and
experiments were conducted at a low cost. Our study supports claims that sex
pheromones are likely to be potent, environmentally benign, species-specific, easy to
apply, and have a low cost of development. (Teeter 1980; Li et a1 2002).

Our results indicate that pheromone-baited traps may compliment integrated sea
lamprey management in the Great Lakes (Smith and Tibbles 1980). Pheromone-baited
traps may reduce the reproductive potential of sea lamprey populations through direct
removal of females from spawning grounds. Additionally, by reducing female
abundance, sterile-male releases may become more effective (Hanson and Manion 1980).
Furthermore, in this study, females were commonly observed swimming directly
upstream into the trap without interacting with the upstream barrier. Therefore,
pheromone-trapping strategies may be conducted on streams that do not have barriers,
while traditional trapping techniques are often conducted in streams with barriers.
Finally, our initial results showed no significant differences between day and night trials;

hence, pheromone trapping may be effective 24 h a day. This may be another advantage

66

to pheromone-based trapping, because traditional trapping techniques are only effective
at night.

Pheromone-baited traps may have broad applications in fisheries management
(Young et al. 2003) because sex pheromones are used by a variety of fish species
(Sveinsson and Hara 1995; Verrneirssen and Scott 2001; Young et al. 2003). Numerous
undesirable fish species are present in North America (Courtenay et al. 1986). Therefore,
sex pheromones may provide an inexpensive and environmentally benign method of fish

control or population estimation (Young et al. 2003).

67

 

ACKNOWLEDGMENTS

We thank Chris Goddard for suggesting this study. Bradley Young assisted with
identification of maturation stages of animals used in this study. Michael Hansen
reviewed an early draft of this manuscript. We thank the staffs of USGS Hammond Bay
Biological Station and USFWS Marquette Biological Station for providing research
facilities, sea lampreys, and equipment. Last, a special thanks to Dolly Trump and Lydia
Lorenz who provided access to their land and Kile Kucher and Jesse Semeyn for helping

conduct this study. The Great Lakes Fishery Commission supported this study.

68

REFERENCES

Applegate, V. C. 1950. Natural history of the sea lamprey in Michigan. U. S.
Department of Interior Fish and Wildlife Service. Special Scientific Report
Fisheries. No. 55.

Bergstedt, R. A., and C. P. Schneider. 1988. Assessment of sea lamprey Petromyzon
marinus predation by recovery of dead lake trout Salvelinus namaycush from
Lake Ontario. Canadian Journal of Fisheries and Aquatic Sciences 45:1406-1410.

Beroza, M., and E. F. Knipling. 1972. Gypsy moth control with the sex attractant
pheromone. Science 177:19-27.

Coble, D. W., R. E. Bruesewitz, T. W. Pratt, and J. W. Scheirer. 1990. Lake trout, sea
lampreys, and overfishing in the upper Great Lakes: A review and reanalysis.
Transactions of the American Fisheries Society 119:985-995.

Courtenay, W.R., D. A. Hensley, J. N. Taylor, and J. A. McCann. 1986. Distribution of
exotic fishes in North America. Pages 675-698 in CH. Hocutt, and ED. Wiley,

editors. The zoogeography of North American freshwater fishes. Wiley, New
York.

Fontaine, M. 1938. La larnproie marine. Sa peche et son importance economique.
Bulletin de la Societe de Oceanographique de France 17:1681-1687.

Great Lake Fishery Commission. 2003. How sea lamprey are controlled? Great Lakes
Fishery Commission. Available: wwwglfcorg/sea lamp/how.asp. (August
2003)

 

Hanson, L. H., and P. J. Manion. 1980. Sterility method of pest control and its potential
role in an integrated sea lamprey Petromyzon marinas control program.
Canadian Journal of Fisheries and Aquatic Sciences 37:2108-2117.

Heinrich, J. W., J. G. Weise, and B. R. Smith. 1980. Changes in biological
characteristics of the sea lamprey Petromyzon marinas as related to lamprey
abundance, prey abundance, and sea lamprey control. Canadian Journal of
Fisheries and Aquatic Sciences 37:1861-1871.

Houston, K. A., and J. R. M. Kelso. 1991. Relation of sea lamprey size and sex-ratio to
salmonid availability in three Great Lakes. Journal of Great Lakes Research 17:
270-280.

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

69

Klar, G. T., and R. J. Young. 2002. Integrated management of sea lampreys in the Great
Lakes 2002. United States Fish and Wildlife Service, and 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. Hanson. 1980. Sea lamprey
control - where to from here? Report of the SLIS control theory task force.
Canadian Journal of Fisheries and Aquatic Sciences 37:2175-2192.

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

Oehlschlager A. C., L. S. Walter, L. Gonzalez, M. Chacon, and R. Andrade. 2003.
Trapping of Phyllophaga elenans with female-produce pheromone. Journal of
Chemical Ecology 29:27-36.

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

Smith, B. R., and J. J. Tibbles. 1980. Sea lamprey Petromyzon marinas in Lakes Huron,
Michigan, and Superior: History of invasion and control, 1936-78. Canadian
Journal of Fisheries and Aquatic Sciences 37:1780—1 801.

Sveinsson, T., and T]. Hara. 1995. Mature males of Arctic Kharr Salvelinus alpinus
release F-type prostaglandins to attract conspecific mature females and stimulate
their spawning behavior. Environmental Biology of Fishes 42:253-266.

Teeter, J .i 1980. Pheromone communication in sea lampreys Petromyzon marinas:
Implications for population management. Canadian Journal of Fisheries and
Aquatic Sciences 37:2123-2132.

Verrneirssen, E. L. M., and A. P. Scott. 2001. Male priming pheromone is present in
bile, as well as urine, of female rainbow trout. Journal of Fish Biology 58:1039-
1045.

Vladykov, V. D. 1949. Quebec lamprey. l.-List of species and their economic
importance. Province of Quebec Department of Fisheries, Contribution. No. 26.

Young, M. K., B. K. Micek, and M. Rathbun. 2003. Probable pheromonal attraction of
sexually mature brook trout to mature male conspecifics. North American Journal
of Fisheries Management 23:276-282.

70

APENDIX C

PERMISSION TO USE PUBLISHED MATERIALS

71

 

Permission to use the following articles

----- Original Message-m-

From: Nicholas S Johnson [mailtozLohnZi32@msu.edu]
Sent: Tuesday, September 27, 2005 1:28 PM

To: joumals@fisheries.org

Subject: Permission to use published materials in thesis

 

American Fisheries Society Journals,

Two of my research papers will be published in the North American Journal of Fisheries
Management. I will be finishing up my Masters program at Michigan State University in
December, and would like to use the published materials in my thesis.

The first paper is already published:

Johnson, N. S., Siefl<es, M. J., and Li W. 2005. Capture of ovulating female sea lampreys
in traps baited with spermiating male sea lampreys. North American Journal of Fisheries
Management 25: 67-72.

The second paper has been accepted for publication and is currently in
press:

Johnson, N. S., Luehring, M. A., Sieflces, M. J ., and Li, W. 2005. Mating pheromone
reception and induced behavior in ovulating female sea lampreys. North American
Journal of Fisheries Management, in press.

Please let me know how I may obtain permission to use these materials in my thesis.
Thanks,

Nicholas Johnson

Masters Student

Michigan State University

Department of Fisheries and Wildlife
517-881-6564

72

From: "Aaron Lemer" <alemer@fisheries.org> [Trust] [Block] To: ioh112132@msu.edu
Date: 28 Sep 2005, 07:58:51 AM
Subject: RE: Permission to use published materials in thesis

 

Dear Mr. Johnson,

The American Fisheries Society grants you permission to use the below-cited
two articles in your thesis.

Aaron

 

Aaron Lerner

Director of Publications
American Fisheries Society
5410 Grosvenor Lane
Bethesda, MD 20814

ph: 301-897-8616, ext. 231
fax: 301-897-5080
www.fisheries.org

73

   

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