FACTORS INFLUENCING PHEROMONE RELEASE BY MALE SEA LAMPREY By Skye Fissette A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Fisheries and Wildlife ─ Master of Science 2017 ABSTRACT FACTORS INFLUENCING PHEROMONE RELEASE BY MALE SEA LAMPREY By Skye Fissette Sea lamprey, Petromyzon marinus, rely on chemical communication to synchronize reproduction. Male sea lamprey release a multicomponent sex pheromone comprised of bile acids that attracts females. 3keto-petromyzonol sulfate (3kPZS) is a main component of this pheromone and induces attraction of sexually mature females. In this thesis, I tested the overall hypothesis that pheromone release is influenced by environmental and social factors. Chapter 1 explores the diel patterns in 3kPZS release and behavioral activity, and the mechanisms involved in 3kPZS release patterns. Male sea lamprey releases larger amounts of 3kPZS at night than during the day. Increased biosynthesis of bile acids at night is likely responsible for this pattern. The regulation of 3kPZS synthesis and release helps synchronize behavior and reproduction in sea lamprey. By pairing increased pheromone release to times of increased reproductive behavior, sea lamprey ensure reproductive success. In Chapter 2, the role of male competition on 3kPZS release patterns was investigated. Male sea lamprey immediately increase 3kPZS release after being exposed to various concentrations of 5-deuterated 3kPZS standard ([2H5] 3kPZS, 5d3kPZS), which was used to simulate male competition. This boost in pheromone signaling intensity allows males to compete for mates by potentially matching or exceeding competitors’ signals. These studies have advanced our understanding of pheromone communication and its role in sea lamprey. Understanding factors influencing pheromone release can help guide the use of pheromones in an integrated control program for sea lamprey in the Great Lakes. ACKNOWLEDGMENTS I would like to thank my advisor Dr. Weiming Li and members of my graduate committee, Drs. Tom Getty and Michael Siefkes for their support and guidance during my studies. Thanks to all members of the Li laboratory for advice and assistance during my master’s program. Special thanks to Dr. Ugo Bussy for conducting all analytical chemistry, and Dr. Yu-Wen Chung Davidson for helping with real time quantitative PCR extraction and analysis. I thank all personnel at the United States Geological Survey/Great Lakes Science Center at Hammond Bay Biological Station in Millersburg MI for being gracious hosts and offering technical assistance. Thanks to the United States Fish and Wildlife Service and Fisheries and Oceans Canada for providing sea lamprey used in all experiments. Special thanks go to all field technicians: Ethan Buchinger, Mike Siemiantkowski, Michelle VanCompernolle, Julia Krohn, Emma Vieregge, Anthony Alvin, and Joseph Riedy whose hard work and dedication made these projects possible and kept me sane. Lastly, I thank the Great Lakes Fishery Commission for funding this research. iii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... vi LIST OF FIGURES .................................................................................................................... vii INTRODUCTION TO THESIS .................................................................................................. 1 PHEROMONE COMMUNICATION IN SEA LAMPREY…………………………………..1 REFERENCES ............................................................................................................................ 5 CHAPTER 1……………………………………………………………………………………...7 DIEL RHYTHMS OF PHEROMONE PRODUCTION AND RELEASE BY MALE SEA LAMPREY .................................................................................................................................... 7 ABSTRACT ................................................................................................................................ 9 INTRODUCTION ..................................................................................................................... 10 METHODS................................................................................................................................ 12 Experimental Animals ........................................................................................................................ 12 Behavioral Observations ..................................................................................................................... 12 Experimental Washings – Diel Pheromone Release ........................................................................... 13 Experimental Washings – Altered Light Cycle .................................................................................. 14 Locomotor Activity and Respiration .................................................................................................. 15 Quantification of Bile Acids in Water Samples .................................................................................. 15 Real-Time Quantitative PCR (RTQ-PCR).......................................................................................... 16 Statistical Analysis .............................................................................................................................. 16 RESULTS.................................................................................................................................. 17 Male Behavior on Nests ...................................................................................................................... 18 Swimming and Respiratory Activity................................................................................................... 20 3kPZS Release Rates – Diel Pheromone Pattern ........................................................................ 22 3kPZS Release Rates – Altered Light Cycle .............................................................................. 24 Transcripts in Liver and Gill Tissue ................................................................................................... 26 DISCUSSION ........................................................................................................................... 28 ACKNOWLEDGMENTS ......................................................................................................... 33 REFERENCES .......................................................................................................................... 34 CHAPTER 2……………………………………………………………………………….........38 PERCEIVED COMPETITION LEADS TO INCREASED PHEROMONE SIGNALING BY MALE SEA LAMPREY ...................................................................................................... 38 ABSTRACT .............................................................................................................................. 40 INTRODUCTION ..................................................................................................................... 41 METHODS................................................................................................................................ 43 Experimental animals .........................................................................................................................43 Experimental Washings ......................................................................................................................43 Quantification of Bile Acids ............................................................................................................... 44 iv Statistical Analysis .............................................................................................................................. 45 RESULTS.................................................................................................................................. 46 3kPZS Release Rates .......................................................................................................................... 46 DISCUSSION ........................................................................................................................... 50 ACKNOWLEDGMENTS ......................................................................................................... 53 REFERENCES .......................................................................................................................... 54 v LIST OF TABLES Table 1-1. Effects of three light cycles on pheromone release in male sea lamprey for 7 days Results of paired t tests on 3kPZS release rates for sea lamprey treated with natural light cycle (DL), continuous darkness (DD) and continuous light (LL) for 7 days prior to experimentation. Number increased indicates the number of sea lamprey increasing 3kPZS release rate from 0000 to 1200, an atypical pattern………………………………............................................................25 Table 1-2. Linear regressions correlating bile acid synthetic enzymes and 3kPZS release rate. Results of linear models correlating 3kPZS release to total transcripts for 10 gene genes in both liver and gill tissue………………………………………………..………………...………27 Table 2-1. Pairwise comparisons of 3kPZS release rates at multiple 5d3kPZS treatment concentrations. Nonparametric pairwise comparisons for treatment-time interactions in the global model of 5d3kPZS treatment concentrations ranging from 5-11 to 5-7 M and a control. All 5k3kPZS treatments were significantly different than the control, but none were significantly different from each other. ATS= ANOVA-type statistic, df=degrees of freedom…………............49 vi LIST OF FIGURES Figure 1-1. Day and night behavioral observations of male sea lamprey on nests in the Cheboygan River. Average number of reproductive behaviors, (A) rock movements and (B) tail fans by individual male sea lamprey on nests. Error bars indicate one standard error of the mean and * indicates significant difference between day and night (p < 0.05)………………………..19 Figure 1-2. Locomotor activity and respiration rate of mature male sea lamprey over a 24 hour time course. (A) Average gill beats per minute and (B) locomotor activity (s) for 6 one hour experimental periods of video recorded mature male sea lamprey (n=6). Shaded area represents night-time hours, non-shaded area represents day-time hours. Error bars indicate one standard error of the mean……………………………………………………………………….21 Figure 1-3. Diel pattern of 3kPZS release by sexually mature male sea lamprey. Average 3kPZS release rate (mg/hr) in water samples from one hour experimental periods conducted on 49 repeatedly measured mature male sea lamprey. Letters indicate significant differences between times based on post-hoc tests with bonferonni correction, same letters are not significantly different from each other. Error bars indicate one standard error of the mean…....23 Figure 1-4. The two major bile acid synthetic pathways used to produce bile acids from cholesterol. Bile acid synthesis starts with HMG-CoA reductase (HMGCR), the rate limiting enzyme that produces cholesterol. The classical pathway uses the rate limiting enzyme CYP7A1 to convert cholesterol into 7α-hydroxycholesterol. HSD3B7 then converts 7αhydroxycholesterol to 7α-hydroxy-4-cholesten-3-one. This is then converted to 7α,12αdihydroxy-4cholsten-3-oone by CYP8B1. In the alternative pathway, CYP27A1 converts cholesterol to 27-hydroxycholesterol. This is converted to 3β, 7α-dihydroxy-5-cholestenoic acid by CYP7B1. In sea lamprey, these are converted into multiple bile acids used as potent sex pheromones through currently unknown mechanisms. Adopted from Li and Chiang (2014)….30 Figure 2-1. Comparisons of 3kPZS release patterns for one 5d3kPZS treatment concentration. Average 3kPZS release rates (mg/hour) in sexually mature male sea lamprey along a 60-minute time course. Base indicates release rate prior to treatment. Males were exposed to a methanol control (diamond) or 5d3kPZS (triangle) treatment. Vertical error bars indicate one standard error of the mean and * indicates significant difference (p<0.05) in release rate between treatments from baseline to the 10-minute time point……………………………..47 Figure 2-2. Comparisons of 3kPZS release patterns at multiple 5d3kPZS treatment toncentrations. Average release rates (mg/hour) of 3kPZS for 6 treatment concentrations across a time course of 60 minutes in sexually mature male sea lamprey. Base indicates release rate prior to treatment. Males were exposed to 5d3kPZS at five concentrations, 5-11 M (diamond), 5-10 M (triangle), 5-9 M (circle), 5-8 M (X), 5-7 M (hollow square), and a methanol control (solid square). Vertical error bars indicate one standard error of the mean……………...…………….48 vii INTRODUCTION TO THESIS PHEROMONE COMMUNICATION IN SEA LAMPREY 1 Chemical communication via pheromones is a widespread form of communication between organisms (Wyatt 2014). Pheromones are compounds released by an individual that initiate behaviors (releaser effect) or may induce physiological or developmental changes (primer effect) in conspecifics (Karlson and Lüscher 1959; Wyatt 2014). Pheromone research has been focused primarily on insects, and only more recently (past 50 years) has research been directed at vertebrates (Symonds and Elgar 2008). When compared to visual and acoustic signals, chemical signals are underrepresented in mate choice literature (Coleman 2009; Johansson and Jones 2007). The sea lamprey’s reliance on chemical signals during reproduction (Buchinger et al. 2015) make it a rare example of a vertebrate species where pheromones have been extensively examined. This makes the sea lamprey a useful system to fill taxonomic and sensory modality gaps in communication systems. The sea lamprey relies on a multicomponent sex pheromone during reproduction (Johnson et al. 2012; Johnson et al. 2006). This has led to intensive studies on sea lamprey pheromone components and what role they play in modulating behavior. A major component of the sex pheromone, 7α,12 α,24-trihydroxy-3-one-5 α -cholan-24-sulfate (3keto-petromyzonol sulfate, 3kPZS) induces both short and long distance upstream movement in sexually mature females (Johnson et al. 2009), affects the locomotor rhythm in ovulating females (Walaszczyk et al. 2016; Walaszczyk et al. 2013), and has priming effects on the neuroendocrine system (Chung-Davidson et al. 2013). 3kPZS is known to induce behavior at a wide range of concentrations, but factors responsible for variation in 3kPZS release are not well understood. Signal variation may be a result of multiple factors (environmental, social, physiological) and the interplay between them. Sea lamprey show nocturnal rhythms in locomotor activity and form aggregations on spawning grounds. Here, I investigated how an environmental factor 2 (light) and social factor (competition) separately affect male release of 3kPZS. Chapter 1, titled, “Diurnal rhythm of pheromone production and release by male sea lamprey”, investigated light cycle as a factor of pheromone release because the nocturnal trend observed during migration becomes less pronounced as the spawning season progresses (Applegate 1950). Experiments confirmed a diel release pattern of 3kPZS that could be disrupted by altering the natural light cycle. I also showed that the bile acid synthetic pathway, locomotor activity, and reproductive behaviors exhibit similar patterns to 3kPZS release. Chapter 1 presents an example of an organism synchronizing a pheromone signal with its behavioral ecology to ensure reproductive success. Aggregation on spawning grounds leads to aggressive behaviors (fights) and competition between males as they defend their nests (Johnson 2015). Chemical communication combined with agonistic behavior offers an opportunity to study how pheromones influence male competition. Chapter 2, titled “Perceived competition leads to increased pheromone signaling in male sea lamprey”, simulated competition using 5-deuterated 3kPZS standard ([2H5] 3kPZS, 5d3kPZS). 5d3kPZS treatments at multiple concentrations led to immediate increases in 3kPZS release. By increasing pheromone release in multiple competitive scenarios, males may better compete for mates by potentially matching or exceeding a competitor’s signal. Chapter 2 provides insights on the effects male competition has in signaling systems. These studies provide evidence that environmental and social factors influence sea lamprey pheromone release. Understanding these factors and their impact on pheromone communication provides further insights into sea lamprey reproductive ecology and highlights the multiple contexts in which pheromone release varies. It is imperative to understand the role of pheromone function in multiple contexts, especially if pheromones are to be implemented as 3 part of an integrative control strategy of invasive sea lamprey in the Great Lakes (Li et al. 2007). Knowing how diel patterns and perceived competition influence pheromone release can help guide effective pheromone use in an integrated control program. For example, in trapping efforts pheromone used as bait would likely need to be higher than the natural release of males to be effective. Knowing the diel pattern of pheromone release can lead to effective daily pumping strategies. Also, using pheromone baited traps may lead to increased pheromone release by males in the system making traps less effective. Understanding this competitive response in males, what concentrations elicit it, and if sea lamprey become habituated can lead to optimal use of pheromone baited traps. 4 REFERENCES 5 REFERENCES Applegate, V. C., 1950. Natural history of the sea lamprey, Petromyzon marinus, in Michigan. US Fish and Wildlife Service. Buchinger, T. J., M. J. Siefkes, B. S. Zielinski, C. O. Brant & W. Li, 2015. Chemical cues and pheromones in the sea lamprey (Petromyzon marinus). Frontiers in zoology 12(1):32. Chung-Davidson, Y.-W., H. Wang, M. J. Siefkes, M. B. Bryan, H. Wu, N. S. Johnson & W. Li, 2013. Pheromonal bile acid 3-ketopetromyzonol sulfate primes the neuroendocrine system in sea lamprey. BMC neuroscience 14(1):11. Coleman, S. W., 2009. Taxonomic and sensory biases in the mate-choice literature: there are far too few studies of chemical and multimodal communication. Acta ethologica 12(1):4548. Johansson, B. G. & T. M. Jones, 2007. The role of chemical communication in mate choice. Biological Reviews 82(2):265-289. Johnson, N. S., Buchinger, T. J., & Li, W. (2015). Reproductive ecology of lampreys Lampreys: biology, conservation and control (pp. 265-303): Springer. Johnson, N., S. S. Yun, T. Buchinger & W. Li, 2012. Multiple functions of a multi‐component mating pheromone in sea lamprey Petromyzon marinus. Journal of fish biology 80(3):538-554. Johnson, N. S., M. A. Luehring, M. J. Siefkes & W. Li, 2006. Mating pheromone reception and induced behavior in ovulating female sea lampreys. North American journal of fisheries management 26(1):88-96. Johnson, N. S., S.-S. Yun, H. T. Thompson, C. O. Brant & W. Li, 2009. A synthesized pheromone induces upstream movement in female sea lamprey and summons them into traps. Proceedings of the National Academy of Sciences 106(4):1021-1026. Karlson, P. & M. Lüscher, 1959. ‘Pheromones’: a new term for a class of biologically active substances. Nature 183(4653):55-56. Li, W., M. Twohey, M. Jones & M. Wagner, 2007. Research to guide use of pheromones to control sea lamprey. Journal of Great Lakes Research 33:70-86. Siefkes, M. J., S. R. Winterstein & W. Li, 2005. Evidence that 3-keto petromyzonol sulphate specifically attracts ovulating female sea lamprey, Petromyzon marinus. Animal behaviour 70(5):1037-1045. 6 Symonds, M. R. & M. A. Elgar, 2008. The evolution of pheromone diversity. Trends in ecology & evolution 23(4):220-228. Walaszczyk, E. J., B. B. Goheen, J. P. Steibel & W. Li, 2016. Differential Effects of Sex Pheromone Compounds on Adult Female Sea Lamprey (Petromyzon marinus) Locomotor Patterns. Journal of biological rhythms 31(3):289-298. Walaszczyk, E. J., N. S. Johnson, J. P. Steibel & W. Li, 2013. Effects of sex pheromones and sexual maturation on locomotor activity in female sea lamprey (Petromyzon marinus). Journal of biological rhythms 28(3):218-226. Wyatt, T. D., 2014. Pheromones and animal behavior: chemical signals and signatures. Cambridge University Press. 7 CHAPTER 1 DIEL RHYTHMS OF PHEROMONE PRODUCTION AND RELEASE BY MALE SEA LAMPREY 8 ABSTRACT Male sea lamprey, Petromyzon marinus, rely on a multicomponent sex pheromone comprised of bile acids that attracts mates. Pairing reproductive behavior with pheromone release may be imperative for reproductive success, especially in a species having a single, reproductive event. Sea lamprey are primarily nocturnal, but it is unknown if pheromone release is consistent with this behavior. In this study, pheromone release under a natural light cycle was quantified by measuring 3keto-petromyzonol sulfate (3kPZS) release at different times of day. 3kPZS release exhibited a diel rhythm with significantly higher release at night than during the day (p < 0.001), and this pattern was disrupted by holding sea lamprey in constant darkness (DD) or constant light (LL) for 7 days. Levels of mRNA for 10 bile acid synthetic enzymes in liver and gill tissue were consistent with the pattern of 3kPZS release, indicating pheromone biosynthesis also exhibits a diel rhythm. The diel trend observed in pheromone production and release is consistent with data showing increased male locomotor activity and reproductive behavior at night. Synchronizing pheromone production and release with behavior and reproductive activity ensures reproduction occurs at optimal times in sea lamprey. 9 INTRODUCTION The biological processes of many organisms follow a circadian rhythm. Circadian rhythms are approximate 24 hour cycles controlled by an endogenous clock and influenced by exogenous factors (zeitgebers) such as light and temperature (Aschoff 1960). This cycle allows animals to respond in changing environmental conditions by altering their behavioral, physiological, or biochemical processes. Regulating such processes could lead to advantages by pairing the appropriate biological response to the appropriate time, especially in the context of reproduction. In many insects, pheromones that are critical for reproduction exhibit diel patterns of release and behavioral activity. The diel timing of pheromone release and response in moths (Baker and Cardé 1979; Foster 2000; Gemeno and Haynes 2000; Rosén 2002; Sower et al. 1970) is important for reliably finding mates to ensure reproductive success and maintain species specificity (Monti et al. 1995; Teal et al. 1978). Pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone synthesis in many moths (Rafaeli and Jurenka 2003) and follows a circadian rhythm in the turnip moth, Agrotis segetum (Rosén 2002; Zavodska et al. 2009). In fish, diel patterns exist for feeding (Boujard and Leatherland 1992), locomotor activity (Reebs 2002) and electric organ discharge (Franchina and Stoddard 1998; Stoddard et al. 2007), but diel patterns of pheromone production and release remain unknown. The sea lamprey, Petromyzon marinus, is an ancestral vertebrate with diel locomotor patterns during parts of its complex life history. During early life, larvae and juveniles display an endogenous, diel locomotor pattern that is eventually lost in constant conditions (Kleerekoper et al. 1961). Adult sea lamprey are nocturnal during migration from oceans to spawning streams but become more active during the day as the spawning season progresses (Applegate 1950; 10 Manion and Hanson 1980). Notably, the male pheromone, 3keto-petromyzonol sulfate (3kPZS), that guides mate search in sea lamprey (Li et al. 2002; Siefkes et al. 2005) modulates the diel locomotor activity of females, likely to synchronize reproductive effort between the sexes (Walaszczyk et al. 2016; Walaszczyk et al. 2013). While the effects of male pheromones on female locomotor patterns are increasingly understood, any diel rhythm in male production and release of pheromones remains undescribed. I determined if pheromone release in sexually mature male sea lamprey displays a diel rhythm and what effect exposure to a constant exogenous factor (light) had on this rhythm. I also investigated two mechanisms potentially playing a role in regulating pheromone release 1) Lamprey regulate activity in the biosynthetic pathway responsible for converting cholesterol into bile acids that comprise the multicomponent pheromone and 2) Increased locomotor activity leads to higher respiration rates and thus an increase in pheromone release across the gill epithelia. Here, I provide evidence of a diel pattern in 3kPZS release that is consistent with activity in the biosynthetic pathway and locomotor activity. 11 METHODS Experimental Animals Sea lamprey were trapped in tributaries of Lakes Huron and Superior by the United States Fish and Wildlife Service and Fisheries and Oceans Canada. Sea lamprey were transported to U.S. Geological Survey laboratory, Hammond Bay Biological Station (HBBS), Millersburg, Michigan. I held sea lamprey in 200-1000 L aerated tanks continually fed with ambient temperature Lake Huron water. All experimental procedures followed protocols approved by the Michigan State University Institutional Animal Care and Use Committee (AUF# 03/14-054-00). Sexually mature males for experiments were collected by holding immature sea lamprey in pvc and mesh cages at the Ocqueoc River, Millersburg, Michigan. Sea lamprey sexually matured in natural river conditions and were checked daily for maturation status using gentle pressure on the abdomen to express milt (Siefkes, 2003). Sexually mature sea lamprey were held at HBBS until experimental use. Behavioral Observations Naturally nesting male and female sea lamprey were observed and sampled from spawning grounds downstream at the Cheboygan River dam, Cheboygan, Michigan during June, 2009. At the Cheboygan River dam, spawning sea lamprey were only found in areas of adequate flow range (0.2-0.6 m/sec). Observations were conducted both the day and night in 2009 (0900 – 1500h and 2300 – 0400h, respectively). Upon locating a nesting male, behavioral observations were recorded for 15 minutes. Standing downstream and to the side of the active nest, an observer recorded water temperature (°C), the number of males and females in the nest, and reproductive behaviors. An additional observer would stand with the recorder and call behaviors as they were observed. Behaviors were recorded individually for males and females and included 12 frequency of rock movements and tail fans, two behaviors associated with reproduction in sea lamprey (Manion and Hanson 1980). The number of males and females per nest were also recorded. Experimental Washings – Diel Pheromone Release Prior to experiments, sexually mature male sea lamprey were held in 200L bonar tanks with aerated Lake Huron water and a clear Plexiglas lid. The clear lid maintained natural light conditions prior to experiments. A Sterilite plastic tub, 61cm x 47cm x 40cm, was modified to be used as an experimental washing chamber. One hole was drilled on each end to connect inflow and outflow hose adapters to ¾” PVC in order to maintain water levels at 17L. These connections were sealed with silicone caulk to prevent leaking. A hole was drilled in the bottom of the tub and fitted with a rubber plug, preventing leaks during experiments. A small slit (2” by 12”) was cut into each lid allowing light to enter while still offering some shade/shelter for sea lamprey. All bins were held in a room with large windows allowing ample natural light inside, and fluorescent lights were never turned on in order to maintain a natural light cycle (~16L:8D). Eight washing chambers were used and fed with aerated, ambient temperature Lake Huron water at ~1L/min. To minimize disturbance, people were only allowed in the room during sampling events. To determine natural pheromone release patterns, 49 sea lamprey from 7 separate groups were sampled repeatedly 6 times a day at 0000, 0400, 0800, 1200, 1600, and 2000. Each group of sea lamprey was randomly assigned a start time and given a three-hour acclimation to avoid any confounding handling effects. An hour before sampling, lake water supply was shut off, drained by removing the bottom plug, and replaced with 14L of deionized (DI) water that was constantly aerated. After one hour, a 50mL water sample was taken, immediately spiked with a 13 5-deuterated 3kPZS standard ([2H5] 3kPZS, 5d3kPZS) (Bridge Organics Inc., Vicksburg, MI) to reach a concentration of 5ng/ml, and then stored in a -20°C freezer. At completion of experiments, sea lamprey were euthanized with an overdose of tricaine methanesulfonate (MS222; Sigma-Aldrich, St. Louis, MO, USA). Liver, gill, and plasma samples were then taken and immediately snap frozen in liquid nitrogen. Blood was collected via cardiac puncture and centrifuged to allow plasma collection. Tissue and plasma samples were held at -80°C until analysis. Experimental Washings – Altered Light Cycle Experiments altering the light cycle follow previous methods with slight modification. Groups of ~7 sea lamprey were randomly assigned to one of three tank conditions: control (natural light cycle, DL), dark (24h of darkness, DD), or light (24h of light, LL). DD tank conditions were achieved using black tarp taped over the entire tank, and LL conditions used pond lights (Laguna POWERGLO PT1578, Laguna, Mansfield, MA, USA). Sea lamprey were held in these tanks for 7 days prior to experimentation and checked twice a day for sea lamprey health. Washing chambers were kept the same as previously described for the DL treatment, but DD bins had no slit on the lid and were covered with multiple coats of black spray paint. LL bins had no slit in the lid and a pond light attached to the interior. Pheromone release patterns were assessed by sampling sea lamprey at 0000 and 1200 (times chosen based on previous results from diel pheromone release experiments). Starting time was randomly chosen and sea lamprey were given a three-hour acclimation. Water and tissues samples were collected as previously described. 14 Locomotor Activity and Respiration Locomotor activity and respiration rate were quantified using video analysis. The same setup and experimental wash chambers for DL were used for this experiment. Infrared lights were attached to the side of the bin, and a Sony Handycam (HDR-XR200V) was placed above for video recording. A temperature logger (HOBO Water Temp Pro v2) was placed in an adjacent bin and recorded temperature every 5 minutes. Sea lamprey were assigned a random start time, given a three hour acclimation period, and then recorded for 24 hours. Locomotor activity and respiration rate were observed for the same six, one hour periods described above. Locomotor activity was defined as consistent movement and was recorded in seconds. Respiration rate was the number of gill beats when the recorder was able to discern them. Factors such as swimming, sea lamprey location, and water disturbance may not allow for visual observation of gill beats, and the duration of time these occurred was not used for calculating beats per minute (BPM). After video analysis, BPM and total swimming activity were calculated for statistical analysis. Quantification of Bile Acids in Water Samples Bile acid concentrations in water were measured using ultra-high performance liquid chromatography tandem mass spectrometry (LC-MSMS) (Xi et al. 2011). Samples were prepared as described in (Brant et al. 2013). 10 mL water samples were spiked with 50 ng of internal standard (5d3kPZS), freeze dried, reconstituted in 100 uL of methanol:water (1:1), transferred to autosampler vials and stored at -20°C in the dark until LC-MSMS analysis. 3kPZS concentrations in water samples were used to calculate 3kPZS release rates for each individual at six time points (0000, 0400, 0800, 1200, 1600, 2000) during the day. 15 Real-Time Quantitative PCR (RTQ-PCR) Methods followed those outlined in (Brant et al. 2013). Briefly, I used TRIzol Reagant (Invitrogen, Carlsbad, CA, USA) to extract total RNA from liver and gill tissues, then treated the RNA with TURBO DNA-free kit (Applied Biosystems, Foster City, CA, USA), and reverse transcribed into cDNA with Moloney Murine Leukemia reverse transcriptase (Invitrogen). RTQPCR was conducted using TaqMan minor groove (Applied Biosystems), and amplification plots were analyzed using Applied Biosystems QuantStudio 6&7 Flex Real-Time PCR System (Applied Biosystems). Synthetic standards were run on each sample plate. Gene transcripts analyzed in gill and liver tissue included hmgcr, cyp7a1, cyp8b1, slc10a1, slc10a2, hsd3b7, sult2b1, sult2a1, bsep, and mdr1 rRNA. Statistical Analysis All statistical analyses were done using R-Studio (RStudio Team (2015). RStudio: Integrated Development for R. Rstudio, Inc., Boston, MA URL http://www.rstudio.com/). To analyze any diel trends, repeated measures ANOVA was used on 3kPZS release rates. Square root transformation was used to meet normality assumptions, and Greenhouse-Geisser correction was used due to violation of sphericity. Post hoc comparisons were conducted using pairwise ttests with bonferonni adjustment to detect differences in 3kPZS release rates between time points. 3kPZS release rates for sea lamprey treated with altered light cycles were log transformed to meet normality assumptions and then analyzed using paired t-tests. Gill beats and swimming activity were analyzed using Friedman tests due to violating assumptions for repeated measures ANOVA. Behavioral observations of nesting males were analyzed using Welch’s t test. Female behaviors were not analyzed because often times multiple females were present on 16 a nest as opposed to only one male. This did not allow for independent observations on individual females. Due to the high variability in 3kPZS release between individuals, and the inability to get repeated measures on tissue samples, RTQ-PCR results were analyzed using linear regression instead of ANOVAs. 3kPZS release rates were used as dependent variables and paired to the appropriate RT-QPCR data (transcripts/ng total RNA) which were used as the independent variable. Each gene transcript was run in an individual model and appropriate transformations were used on the independent variable when deemed necessary by analyzing model diagnostics (histograms, residuals vs. fitted values, and qq plots). 17 RESULTS Male Behavior on Nests Welch’s t test indicate a significant increase in tail fans between day and night t(35.7) = -2.47, p = 0.02 but no significant difference in the amount of rock moves t(40.7) = -1.65, p = 0.11 (Figure 1-1). While rock moves were not significant between day and night, nest observations indicated an increase of reproductive behaviors at night, when males tend to be more active. 18 Figure 1-1. Day and night behavioral observations of male sea lamprey on nests in the Cheboygan River. Average number of reproductive behaviors, (A) rock movements and (B) tail fans by individual male sea lamprey on nests. Error bars indicate +/- 1 standard error of the mean and * indicates significant difference between day and night (p < 0.05). 19 Swimming and Respiratory Activity Friedman test indicated significant effects of time on swimming activity X2(5) = 16.03 , p < 0.01 and respiration rate X2(5) = 12.95 , p = 0.02 on video recorded sea lamprey (Figure 12). Post hoc tests could not detect differences between times due to low sample sizes (n=6) for both respiratory and swimming activity. Male sea lamprey had elevated locomotor activity and respiration rate at night. Temperatures averaged 20.6°C and ranged from 17.8°C to 22.2°C. These are within the natural range seen in streams during sea lamprey reproduction. 20 Figure 1-2. Locomotor activity and respiration rate of mature male sea lamprey over a 24 hour time course. (A) Average gill beats per minute and (B) locomotor activity (s) for 6 one hour experimental periods of video recorded mature male sea lamprey (n=6). Shaded area represents night-time hours, non-shaded area represents day-time hours. Error bars indicate one standard error of the mean 21 3kPZS Release Rates – Diel Pheromone Pattern For the repeated measures ANOVA, Mauchly’s test indicated the assumption of sphericity was violated X2(14) = 51.4, p < 0.001, therefore degrees of freedom were corrected using Greehouse-Geisser estimates of sphericity (ε=0.70). The data showed a significant effect of time on 3kPZS release F(3.5, 33.6) = 27.5, p < 0.001. Post-hoc comparisons indicated a diel pattern of 3kPZS release (Figure 1-3). 3kPZS was released at significantly higher rates during the night (0000, 0400 h) compared to the day (0800, 1200, and 1600 h). 3kPZS release at 2000h, a time nearing sunset, was also significantly higher than day-time hours. 22 Figure 1-3. Diel pattern of 3kPZS release by sexually mature male sea lamprey. Average 3kPZS release rate (mg/hr) in water samples from one hour experimental periods conducted on 49 repeatedly measured mature male sea lamprey. Letters indicate significant differences between times based on post-hoc tests with bonferonni correction, same letters are not significantly different from each other. Error bars indicate one standard error of the mean. 23 3kPZS Release Rates – Altered Light Cycle Paired t-tests conducted on log transformed data indicate a significant decrease in 3kPZS release rate between 0000 and 1200 h for the DL group (t(15)=3.51, p=0.003) and no significant decrease for the DD (t(17)=1.98, p=0.06) or LL (t(18)=1.05, p=0.31) groups. Holding sea lamprey in DD or LL conditions for 7 days significantly disrupted the natural release pattern of 3kPZS as a significant decrease in release rate was no longer seen between 0000 and 1200 h (Table 1-1). 24 Table 1-1. Effects of three light cycles on pheromone release in male sea lamprey for 7 days Treatment DL DD LL n 17 18 19 Night Release Rate (mg/hr) Mean ± Std. Error 0.31 ± 0.05 0.35 ± 0.06 0.32 ± 0.06 Day Release Rate (mg/hr) Mean ± Std. Error 0.21 ± 0.05 0.23 ± 0.05 0.26 ±0.05 Number Increased 3 6 7 p value 0.003 0.06 0.31 Results of paired t tests on 3kPZS release rates for sea lamprey treated with natural light cycle (DL), continuous darkness (DD) and continuous light (LL) for 7 days prior to experimentation. Number increased indicates the number of sea lamprey increasing 3kPZS release rate from 0000 to 1200, an atypical pattern 25 Transcripts in Liver and Gill Tissue Linear regression showed significant correlations between 3kPZS release and bile acid synthetic enzymes in both liver and gill tissue (Table 1-2). In liver tissue, transcripts of the genes encoding the rate limiting enzyme for cholesterol synthesis (hmgcr), the rate limiting enzyme for bile acid synthesis (cyp7a1), two bile acid synthetic enzymes (cyp8b1, cyps7a1), and two bile acid transporters (slc10a1, slc10a2) were significantly correlated with 3kPZS release (p<0.05). In gill, transcripts of hmgcr, cyp7a1, and cyp8b1 were significantly correlated to 3kPZS release (p<0.05). 26 Table 1-2. Linear regressions correlating bile acid synthetic enzymes and 3kPZS release rate Gene Transcript-Tissue Function Transformation β R2 p-value HMGCR - Liver rate limiting enzyme for cholesterol synthesis log 2.8 0.20 < 0.01 cyp8b1 - Liver bile acid synthetic enzyme square root 0.006 0.39 < 0.001 cyp27a1 - Liver bile acid synthetic enzyme none 1.13 E-7 0.15 < 0.01 cyp7a1 - Liver rate limiting enzyme for bile acid synthesis square root 0.0007 0.38 < 0.001 hsd3b7 - Liver 3-keto group modification log -1.29 0.001 0.84 sult2a1 -Liver bile acid sulfotransferase log 1.7 0.006 0.6 slc10a1 - Liver bile acid transporter, into hepatocyte log 23.1 0.44 < 0.001 slc10a2 - Liver bile acid transporter, into cell ? log 8.14 0.11 0.03 bsep - Liver bile acid transporter, out of hepatocyte log 2.19 0.01 0.44 mdr1 - Liver organic acid transporter, out of hepatocyte log 2.31 0.02 0.346 HMGCR - Gill rate limiting enzyme for cholesterol synthesis log 7.02 0.31 < 0.001 cyp8b1 - Gill bile acid synthetic enzyme log 12.2 0.12 0.02 cyp27a1 - Gill bile acid synthetic enzyme none 3.12 E-6 0.02 0.34 cyp7a1 - Gill rate limiting enzyme for bile acid synthesis log 20.1 0.17 < 0.01 hsd3b7 - Gill 3-keto group modification none 5.43 E-6 0.01 0.45 sult2a1 -Gill bile acid sulfotransferase none -3.52 E-7 0.006 0.61 slc10a1 - Gill bile acid transporter, into hepatocyte log -3.50 0.01 0.48 slc10a2 - Gill bile acid transporter, into cell log 1.95 0.004 0.67 bsep - Gill bile acid transporter, out of hepatocyte log 2.40 0.004 0.68 mdr1 - Gill organic acid transporter, out of hepatocyte square root 0.01 0.003 0.74 Results of linear models correlating 3kPZS release to total transcripts for 10 gene genes in both liver and gill tissue 27 DISCUSSION Our results support the hypothesis that a circadian rhythm modulates pheromone production and release by male sea lamprey. Pheromone release rates followed a diel pattern that was disrupted during constant exposure to an altered environmental stimulus (light) for 7 days. Endogenous control, demonstrated by a free running period or phase shift in diel pattern, was not investigated for pheromone release but has been shown to occur in lamprey locomotor activity (Kleerekoper et al. 1961) and melatonin release (Samejima et al. 2000; Samejima et al. 1997). In each case, this period was gradually lost under constant conditions mirroring our results and suggests pheromone release would display a free running period. Observed patterns in pheromone release appear to be regulated by the bile acid synthetic pathway. Synchronization of reproductive behavior is especially important in sea lamprey due to a single, reproductive event and lack of energy resources caused by feeding cessation at the reproductive stage (William and Beamish 1979). Previous research has shown pheromones modulate locomotor rhythmicity in female sea lamprey, increasing activity during the day in ovulated females (Walaszczyk et al. 2016; Walaszczyk et al. 2013), likely to synchronize behavior with males who become more active during the day. While male activity does increase during the day, data presented here shows sexually mature males were still more active at night, a trend also seen in river lamprey, Lampetra fluviatis (Sjöberg 1974). There is also a pattern of increased male reproductive behaviors at night. Pairing increased activity and reproductive behavior with elevated pheromone signals could be essential in synchronizing male and female reproduction at optimal times, especially given the knowledge that females prefer higher concentrations of 3kPZS (Johnson et al. 2009). Combining these factors could lead to increased chances of spawning events and improved reproductive fitness. 28 Male sea lamprey release pheromone across the gill epithelia (Siefkes et al. 2005) and upon sexual maturation in sea lamprey, there is a dramatic upregulation of expression in cyp7a1, a rate limiting enzyme for bile acid synthesis (Russell and Setchell 1992) and in cyp27a1 and cyp8b1, in the liver (Brant et al. 2013; Yeh et al. 2012). These upregulations have evolved to allow increased synthesis (Figure 1-4, adapted from (Li and Chiang 2014)) and secretion of bile acids, allowing them to have the added function as a sex pheromone. RTQ-PCR results from liver show significant, positive correlations between 3kPZS release and the amount of transcribed mRNA for genes encoding enzymes involved in cholesterol synthesis (hmgcr), bile acid synthesis (cyp7a1, cyp8b1, cyp27a1) and bile acid transport (slc10a1, slc10a2). In gill tissue, significant correlations are observed for hmgcr, cyp7a1, and cyp8b1 transcripts. Currently, there is no evidence to support bile acid synthesis in gill tissue of fish, but conversion of bile acids (PZS to 3kPZS) has been suggested in sea lamprey (Brant 2013). The presence of HMGCR and bile acid synthetic enzymes in sea lamprey gills combined with significant correlations to 3kPZS release suggests the possibility of de novo bile acid synthesis in gill tissue, a process seen in parasitic sea lamprey intestine (Yeh et al. 2012). Regulation of the biosynthetic pathway is consistent with the pattern of 3kPZS release. By controlling this regulation, male sea lamprey have evolved a diel pattern of pheromone release in order to synchronize reproduction. 29 Figure 1-4. The two major bile acid synthetic pathways used to produce bile acids from cholesterol. Bile acid synthesis starts with HMG-CoA reductase (HMGCR), the rate limiting enzyme that produces cholesterol. The classical pathway uses the rate limiting enzyme CYP7A1 to convert cholesterol into 7α-hydroxycholesterol. HSD3B7 then converts 7αhydroxycholesterol to 7α-hydroxy-4-cholesten-3-one. This is then converted to 7α,12αdihydroxy-4cholsten-3-oone by CYP8B1. In the alternative pathway, CYP27A1 converts cholesterol to 27-hydroxycholesterol. This is converted to 3β, 7α-dihydroxy-5-cholestenoic acid by CYP7B1. In sea lamprey, these are converted into multiple bile acids used as potent sex pheromones through currently unknown mechanisms. Adopted from Li and Chiang (2014). 30 A diel pattern in sea lamprey locomotor behavior and subsequently respiration activity may play a role in increased pheromone release. Swimming activity and respiration rate follow a similar trend seen in 3kPZS release at times ranging from 0000 to 1600, being high at night and dropping as the day progresses. However, activity and respiration rate still remains very low at 2000, a time when 3kPZS release is near its highest. This difference suggests swimming activity and its effect on respiration may play some role in pheromone release, but it is not likely the main reason behind the diel trend. One possible explanation for observed differences in release and respiration at 2000 may be the small sample size (n=6). Future experiments should be aimed at understanding how respiration rate affects pheromone release. The mechanism responsible for regulating the pattern seen in the biosynthetic pathway and pheromone release remains undescribed. One explanation may be linked to the hormone, melatonin, which is released from the pineal complex. The pineal organ in lamprey is photosensitive (Morita and Dodt 1971; Tamotsu and Morita 1986) and is suggested to control circadian locomotor activity (Morita et al. 1992) but see (Samejima et al. 2000). Arctic lamprey, Lethenteron camtschaticum, have an endogenous oscillator in the pineal gland responsible for circadian rhythms of melatonin release (Samejima et al. 2000; Samejima et al. 1997). Melatonin has been shown to play a role in circadian rhythms in lower vertebrates and may be responsible for synchronizing internal cycles to the environment (Underwood 1989). Daily rhythms in vertebrates are also influenced by feedback loops consisting of clock genes (Zhang and Kay 2010). In the bile acid synthetic pathway, expression of cyp7a1 is regulated by both positive and negative clock genes (Lavery and Schibler 1993; Noshiro et al. 2004), but regulation of rhythms in organs such as the liver are also influenced by feeding cycles (Damiola et al. 2000; Zhang et 31 al. 2011). Because the adult sea lamprey used in our study were no longer feeding, clock genes may play a role in regulating the biosynthetic pathway and thus pheromone release. In conclusion, I show a clear diel pattern of pheromone release from sexually mature male sea lamprey and provide evidence that this pattern can be modulated by altering the light cycle. Amounts of mRNA for rate limiting enzymes in the bile acid synthetic pathway and data on respiration and swimming activity are consistent with the diel release of 3kPZS. This is consistent with the reproductive biology of sea lamprey, and the need to synchronize behavior through chemical communication for reproductive success. 32 ACKNOWLEDGMENTS I am grateful to the personnel of the U.S Geological Survey Hammond Bay Biological Station for the use of facilities and technical support. The U.S Fish and Wildlife Service and Fisheries and Oceans Canada provided sea lamprey used in experiments. Research technicians Ethan Buchinger, Mike Siemiantkowski, Michelle VanCompernolle, Julia Krohn, Emma Vieregge, Anthony Alvin, and Joseph Riedy provided assistance on experiments and video analysis. 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Differential Effects of Sex Pheromone Compounds on Adult Female Sea Lamprey (Petromyzon marinus) Locomotor Patterns. Journal of biological rhythms 31(3):289-298. Walaszczyk, E. J., N. S. Johnson, J. P. Steibel & W. Li, 2013. Effects of sex pheromones and sexual maturation on locomotor activity in female sea lamprey (Petromyzon marinus). Journal of biological rhythms 28(3):218-226. William, F. & H. Beamish, 1979. Migration and spawning energetics of the anadromous sea lamprey, Petromyzon marinus. Environmental Biology of Fishes 4(1):3-7. 37 Xi, X., N. S. Johnson, C. O. Brant, S.-S. Yun, K. L. Chambers, A. D. Jones & W. Li, 2011. Quantification of a male sea lamprey pheromone in tributaries of Laurentian Great Lakes by liquid chromatography–tandem mass spectrometry. Environmental science & technology 45(15):6437-6443. Yeh, C.-Y., Y.-W. Chung-Davidson, H. Wang, K. Li & W. Li, 2012. Intestinal synthesis and secretion of bile salts as an adaptation to developmental biliary atresia in the sea lamprey. Proceedings of the National Academy of Sciences 109(28):11419-11424. Zavodska, R., G. von Wowern, C. Löfstedt, W. Rosen & I. Sauman, 2009. The release of a pheromonotropic neuropeptide, PBAN, in the turnip moth Agrotis segetum, exhibits a circadian rhythm. Journal of insect physiology 55(5):435-440. Zhang, E. E. & S. A. Kay, 2010. Clocks not winding down: unravelling circadian networks. Nature reviews Molecular cell biology 11(11):764-776. Zhang, Y.-K. J., G. L. Guo & C. D. Klaassen, 2011. Diurnal variations of mouse plasma and hepatic bile acid concentrations as well as expression of biosynthetic enzymes and transporters. PloS one 6(2):e16683. 38 CHAPTER 2 PERCEIVED COMPETITION LEADS TO INCREASED PHEROMONE SIGNALING BY MALE SEA LAMPREY 39 ABSTRACT Males of many animal species produce signals that aggregate conspecifics and attract mates. Males can acquire information and alter signals in response to the presence of competitors. Competition has exerted varying pressures on the evolution of signal systems. Relative to visual and acoustic signals, less is known about how chemical signals affect competition. The sea lamprey, Petromyzon marinus, relies on chemical communication for reproduction, offering a useful system to study the role of chemical signals on male-male competition. Male sea lamprey aggregate on spawning grounds where individuals build a nest and signal to females using sex pheromones. A major component, 3keto-petromyzonol sulfate (3kPZS), induces short and long distance upstream movement of sexually mature females. Here, I examined the male response to mate competition, using 3kPZS to simulate the presence of mature males. The simulated competition led to an immediate increase in 3kPZS release rate in two separate experiments (p = 0.02 and x̅ = 91%, p = 0.01 and x̅ = 315%) within ten minutes followed by a reduction to baseline levels over the course of an hour. I conclude by increasing 3kPZS release, males may improve the odds of matching or possibly exceeding competitors’ signals, allowing them to better compete for mates. 40 INTRODUCTION The diversity of elaborate male traits has perplexed biologists since Darwin (Darwin 1871). Accumulating evidence shows both intra- and inter-sexual selection shape the evolution of male traits and signaling strategies in many taxa (Andersson 1994). Variation in male signals combined with female choice can influence individual mating success, driving the evolution of signaling systems (Basolo 1990; Kirkpatrick 1987; Moore et al. 2001). In communication networks, bystanders within the range of a signal may eavesdrop and obtain beneficial information used to modulate their own signaling or behavior strategies, putting added selection pressure on signaling systems. (Earley 2010; McGregor 2005; McGregor and Peake 2000). In reproductive aggregations, it may be necessary to match or exceed a competitor’s signal in order to better compete for mates, especially when a preference is shown for critical signal attributes. Well documented examples of signal changes in competitive scenarios are established in a variety of taxa. Several species of male anurans have the ability to significantly alter the dominant frequency of acoustic signals during social interactions with competing males (Bee and Perrill 1996; Lopez et al. 1988; Wagner 1989). Male lesser wax moths, Achroia grisella, increase ultrasonic signal rates when in close proximity (<40cm) to signaling males (Jia et al. 2001). Male lekking fruit flies, Drosphila grimshawi, adjust pheromone deposition in response to competitors’ pheromone deposits (Widemo and Johansson 2006), and male house mice, Mus domesticus, increase scent marking rates when detecting marks from competitors (Humphries et al. 1999; Hurst 1990). The evolution and selection pressures acting on chemical communication is an underrepresented area of signal research (Coleman 2009; Symonds and 41 Elgar 2008), and the role pheromones play on male-male competition are not well described (Wyatt 2014). Reliance on chemical communication (Buchinger et al. 2015) along with male competition on spawning grounds (Johnson et al. 2015) make the sea lamprey, Petromyzon marinus, an excellent model to study the role pheromones have on male-male competitive interactions in a basal vertebrate. The sea lamprey is an ancestral, jawless vertebrate where olfaction is the dominant sensory modality used during reproduction (Johnson et al. 2006; Teeter 1980; Vrieze et al. 2010). When sexually mature, males aggregate on spawning grounds, build a nest, and rely on sex pheromones comprised of bile acids that attract females (Li et al. 2002; Siefkes et al. 2005). A major component of the sex pheromone, 7α,12 α,24-trihydroxy-3-one-5 α -cholan-24-sulfate (3keto-petromyzonol sulfate, 3kPZS), is released through the gills (Siefkes et al. 2005), and to which the behavioral response of females is well characterized (Johnson et al. 2009). Here, I investigated whether male sea lamprey altered pheromone release rate upon perceiving competition, and whether males have a measured response to the perceived strength of competition. I provide evidence that male sea lamprey increase their pheromone signal to simulated competition at multiple treatment concentrations. 42 METHODS Experimental animals All handling and experimental procedures involving sea lamprey followed protocols approved by the Michigan State University Institutional Animal Care and Use Committee (AUF# 03/14-054-00). Sea lamprey were trapped in tributaries of Lakes Huron and Superior by the United States Fish and Wildlife Service and Fisheries and Oceans Canada. Sea lamprey were transported to U.S. Geological Survey laboratory, Hammond Bay Biological Station (HBBS), Millersburg, Michigan where they were held in 200-1000 L aerated tanks continually fed with ambient temperature Lake Huron water. Sexually mature males for experiments were collected by holding immature lamprey in cages at the Ocqueoc River, Millersburg, Michigan. These sea lamprey were allowed to mature in natural river conditions and checked daily for maturation using methods by Siefkes et al. (2003). Sexually mature sea lamprey were brought back to HBBS and held until experimental use. Experimental Washings Water samples conditioned with sexually mature sea lamprey were collected for quantification of bile salts. A five-gallon bucket was modified to be used as an experimental wash chamber. A hole was drilled in the bottom for a rubber plug. This prevented leaks during experimental trials. One hole was drilled on the side connecting a ¾” pvc elbow to an outflow, keeping water levels a consistent 5 L during the acclimation period. Prior to experiments, 15 to 20 sexually mature males were held for 12-14 hours in an aerated 200 L bonar tank continually fed with Lake Huron Water held at 16-18°C using a 43 temperature control unit. Individual sea lamprey were removed from this tank and transferred into a wash chamber for an hour acclimation period to remove them from trace smells of other males. Each bucket received aerated Lake Huron water at a rate of ~500 ml/min during acclimation. After one hour, water was drained by pulling the plug on the bottom of the bucket and replaced with 3 L of deionized (DI) water that was constantly aerated. After 30 minutes, a 50 ml water sample was taken and immediately spiked with a 5-deuterated 3kPZS standard ([2H5] 3kPZS, 5d3kPZS) (Bridge Organics Inc., Vicksburg, MI) to reach a concentration of 1ng/ml. This thirty-minute sample served as baseline measure for pheromone release prior to treatment. Immediately following this baseline sampling event, a treatment was administered and swirled around to ensure thorough mixing. Two different treatment regimens were conducted, one treatment included 5d3kPZS (applied at 5x10-10 molar) and 1mL of 50% methanol used as a control (Experiment 1). The other included 5d3kPZS applied at one of five concentrations ranging from 5x10-11 to 5x10-7 molar and a 1mL 50% methanol control (Experiment 2). Subsequent water samples were taken 10, 30, and 60 minutes after treatment application. All control treatment samples were spiked with a 5d3kPZS as an internal standard, whereas 5d3kPZS treatment samples were spiked with 11-deuterated 3kPZS ([2H11] 3kPZS, 11d3kPZS) (Bridge Organics Inc., Vicksburg, MI) as a standard. 10 mL subsamples were taken from each 50 mL sample, and both were immediately transferred to a -20°C freezer. All treatments were randomized within each block of experiments. Experiment 1 had n=20 for each treatment group and experiment 2 had n=13 for each treatment group. Quantification of Bile Acids Sea lamprey bile acid concentrations in water were measured by ultra-high performance liquid chromatography tandem mass spectrometry (LC-MSMS) (Xi et al. 2011). Samples were 44 prepared as previously described (Brant et al. 2013). Briefly, 10 mL water samples were spiked with 10 ng of internal standard (3kPZS-d5), freeze dried, reconstituted in 100 uL of methanol:water (1:1), transferred to an autosampler vial and stored at -20°C until LC-MSMS analysis. 3kPZS concentrations in water samples were used to calculate average release rates for each individual at the four time points between sampling intervals. Statistical Analysis All statistical analyses were done using R-Studio (RStudio Team (2015) RStudio: Integrated Development for R. Rstudio, Inc., Boston, MA URL http://www.rstudio.com/). Average 3kPZS release rates in water samples were analyzed using the nparLD package in R, allowing non parametric analysis on longitudinal data. Multiple comparisons using F1-LD-F1 models with bonferonni adjustment were used to determine differences between the baseline release rate and each sampling event for experiment 1. Because I decided to focus on effects the treatment concentration played on pheromone release pattern for experiment 2, pairwise comparisons were retrieved using the pair.comparison function in the nparLD package to determine differences in release patterns of the global model. 45 RESULTS 3kPZS Release Rates A global comparison (F1-LD-F1 model) with one whole plot factor (treatment) and one sub-plot factor (time) yielded significant effects of treatment and time interactions for 3kPZS release rates in experiment 1 (ANOVA-type statistic, ATS = 3.74, df = 2.24 p =.02) (Figure 2-1) and experiment 2 (ANOVA-type statistic, ATS = 2.27, df = 9.97, p =.01) (Figure 2-2) indicating different release patterns of 3kPZS. In experiment 1, an increase in 3kPZS release was observed at 10 minutes when compared to the baseline (ANOVA-type statistic, ATS = 22.1, df = 1, p = < 0.001), however the release rates at 30 minute (ANOVA-type statistic, ATS = 1.00, df = 1, p = 0.93) and 60 minute (ANOVA-type statistic, ATS = 0.02, df = 1, p = 1) minute time points were not different than the baseline between treatments. In experiment 2, each 5d3kPZS treatment concentration resulted in an increase of 3kPZS release compared to control, but no significant differences were found when comparing between 5d3kPZS treatments (Table 2-1). 46 Figure 2-1. Comparisons of 3kPZS Release Patterns for one 5d3kPZS Treatment Concentration. Average 3kPZS release rates (mg/hour) in sexually mature male sea lamprey along a 60-minute time course. Base indicates release rate prior to treatment. Males were exposed to a methanol control (diamond) or 5d3kPZS (triangle) treatment. Vertical error bars indicate one standard error of the mean and * indicates significant difference (p<0.05) in release rate between treatments from baseline to the 10-minute time point 47 Figure 2-2. Comparisons of 3kPZS Release Patterns at multiple 5d3kPZS Treatment Concentrations. Average release rates (mg/hour) of 3kPZS for 6 treatment concentrations across a time course of 60 minutes in sexually mature male sea lamprey. Base indicates release rate prior to treatment. Males were exposed to 5d3kPZS at five concentrations, 5-11 M (diamond), 5-10 M (triangle), 5-9 M (circle), 5-8 M (X), 5-7 M (hollow square), and a methanol control (solid square). Vertical error bars indicate one standard error of the mean. 48 Table 2-1. Pairwise comparisons of 3kPZS Release Rates for Multiple 5d3kPZS Treatment Concentrations Comparison 5 M vs. Control ATS 4.16 df 2.51 p-value 0.009 5-10 M vs. Control -11 4.73 2.63 0.004 -9 3.96 2.43 0.01 -8 7.86 2.73 < 0.001 -7 5 M vs. Control 5 M vs. Control 5 M vs. Control 5-11 M vs. 5-10 M 5-11 M vs. 5-9 M 2.99 0.84 2.34 2.39 0.04 0.45 0.86 1.94 0.45 5-11 M vs. 5-8 M 0.83 1.94 0.43 -7 0.87 2.20 0.43 -10 -9 0.09 2.22 0.93 -10 -8 0.98 2.77 0.39 -10 -7 5 M vs. 5 M 1.71 2.77 0.16 5-9 M vs. 5-8 M 1.53 2.81 0.21 5-9 M vs. 5-7 M 1.76 2.39 0.16 2.01 2.31 0.13 5 -11 M vs. 5 M 5 M vs. 5 M 5 M vs. 5 M -8 -7 5 M vs. 5 M Nonparametric pairwise comparisons for treatment-time interactions in the global model of 5d3kPZS treatment concentrations ranging from 5-11 to 5-7 M and a control. All 5k3kPZS treatments were significantly different than the control, but none were significantly different from each other. ATS= ANOVA-type statistic, df=degrees of freedom. 49 DISCUSSION Increases in pheromone release patterns by male sea lamprey support the hypothesis that males alter their signal upon perceiving competition from other males. The immediate spike of 3kPZS release rates in both experiments indicate a boost in pheromone signalling intensity. The capability to rapidly increase a pheromone signal is adaptive in the framework of sea lamprey reproductive biology. Female sea lamprey show a preference for higher 3kPZS concentrations when given a direct choice (Johnson et al. 2009). Increasing pheromone signal intensity allows males to potentially match or exceed nearby competitors’ signals thus gaining better access to potential mates. In our experiments, increases in 3kPZS release rates averaged 91% (experiment 1) and 315% (experiment 2) resulting in concentration changes biologically relevant to females (Johnson 2009). The evolution of this signal increase is beneficial in a system where males are in close proximity on spawning grounds and females show preference for the amount of a critical signal trait. The male response to different concentrations parallels female behavioral response to 3kPZS. Female response remains the same at distances of 70m and 650m to pheromone concentrations varying from 10-14 M to 10-10 M (Johnson et al. 2009). A concentration below 1014 M may still illicit increased release, but this is unlikely. After the baseline period, average 3kPZS concentration for all lamprey was 34 ng/ml and calculates out to a concentration increase of 3.8-11 M 3kPZS per second. This suggests a treatment concentration lower than this would likely not elicit the observed increase. Different concentrations eliciting the same response allows male lamprey to modify their own signal within a wide range of biological conditions such as male abundance and streamflow that can vary both within and across spawning streams. This response also fits well within the context of the large variation observed in pheromone 50 release across individuals. Certain males may not be able to compete effectively when encountering a large signal; therefore, the best option may be releasing a large amount of stored pheromone in an effort to do the best their physiology allows. Release rate increase is only observed in the first 10 minutes following 5d3kPZS treatment exposure. This is likely explained by an overall limitation in resources due to an increase in costs associated with boosting pheromone production and release and the limited energy stores available during reproduction after feeding cessation at the onset of the reproductive migration (William and Beamish 1979). Costs of pheromone production are unknown in sea lamprey, but a similar system appears in lesser wax moths, which increase their signal rate only for a short duration (5-10 min) due to metabolic costs and limited energy resources caused by a lack of adult feeding (Jia et al. 2001). Because I was only able to measure average pheromone release in this 10-minute time frame, it is possible there is not a constant increase in release rate but rather a large release for a few minutes or less. It is unlikely the bile acid synthetic pathway, subsequent secretion into circulation, and transport to the gills for release, in concert are being upregulated this quickly. It is reasonable to conclude males release a large quantity of previously synthesized and stored 3kPZS. Our results also indicate a pulse of 3kPZS is needed to illicit the observed response, as an individual sea lamprey’s own, consistent 3kPZS release into the bucket was not enough. This response would be potentially beneficial when a large number of new, sexually mature males begin signaling or when a male comes within close proximity of another male(s). These situations expose males to increases in pheromone concentrations, where they adapt their own signal to better compete. At this time, it is not determined if sea lamprey have the ability to repeatedly increase their signal after multiple 51 exposures to a pheromone treatment, or if there is a potential lag time needed before an individual can boost its pheromone signal again. In conclusion, I give evidence showing male sea lamprey alter their pheromone signal in response to simulated competition. This likely evolved to increase competitive ability of males through use of chemical communication. Further investigations are needed to define the mechanisms responsible for this signal boost and the cost of pheromone production. 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