PLACE IN RETURN BOX to remove this checkout from your record. i To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K'IProj/AccaPresICiRC/DaIeDue,indd __..E h, , - *~+A—- ,..._..._-_....._. ~ 4-—-.,. A,” _ 4 -.. - .-..._. _....._...-.‘..._.~.. -HMM ._- IN-STREAM BEHAVIORAL RESPONSES OF FEMALE SEA LAMPREYS TO PHEROMONE COMPONENTS By Nicholas S. Johnson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Fisheries and Wildlife 2008 ABSTRACT IN-STREAM BEHAVIORAL RESPONSES OF FEMALE SEA LAMPREYS TO PHEROMONE COMPONENTS By Nicholas S. Johnson Interference with sea lamprey (Petromyzon marinus) chemical communication may Offer effective and benign methods to manage their populations in the Great Lakes where they are destructive predators of large fishes. Previous studies showed that a mating pheromone, putatively consisting of 3-keto petromyzonol sulfate (3kPZS) and 3- keto allocholic acid (3kACA), is excreted by spermiated males and directs ovulated females to spawning nests. Synthesized 3kPZS elicits preference responses in ovulated females in a natural stream, but it is unknown if 3kPZS is the only component of the mating pheromone and whether female responses to synthesized 3kPZS are sufficiently strong to merit its use in management. I hypothesized that 3kPZS would elicit robust upstream movements in ovulated females, directing them into traps and luring them away from natural male odorants. In this dissertation, in-stream behavioral tests showed that ovulated females responded with robust upstream movement directly to the source of synthesized 3kPZS concentrations ranging from 10'10 to 10'14 molar (M) and in diverse stream conditions. Ovulated female responses to 3kPZS are sufficiently strong to support utility in management where nearly 50% of ovulated females were captured in 3kPZS- baited traps and high concentrations of 3kPZS lured females away from and disrupted orientation to a natural pheromone source. Given that 3kPZS induced ovulated females to migrate upstream, and that it was recently discovered that sea lamprey larvae release 3kPZS, responses of pre-ovulatory females to 3kPZS were re-evaluated at night. Contrary to previous studies, 3kPZS induced strong preference responses in pre- ovulatory females not differing from that elicited by larval migratory pheromone over long distances. 3kPZS may not function specifically as a mating pheromone component, but as a pheromone component that induces directed migration in spawning-phase females regardless of maturity. Therefore, 3kPZS may have greater impacts on sea lamprey management than previously conceived, as it could potentially be used to modify the behavior and distribution of females during the entire migratory period, in addition to the spawning period. However, when 3kPZS and natural mating pheromone were compared directly, it was clear that additional pheromone components were released by males to retain females on nests and induce mating behaviors. 3kACA, previously hypothesized to retain ovulated females on nests, was extensively tested in streams, but it did not modify ovulated female behavior. A new in-stream bioassay was developed to confirm that XAD7HP resin extracted unidentified pheromone components that induced mating behaviors, and that spermiated males release all behaviorally active pheromone components through the head region. Future identification of additional pheromone components from XAD7HP extract using the in-stream bioassay will enable mating pheromone components to be fully characterized and all potential mating pheromone- based management tactics to be realized. Synthesized 3kPZS must be tested in management contexts containing wild lampreys to confirm its utility for sea lamprey control. To my dad who told me to work hard and be happy... iv ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Weiming Li and the members of my graduate committee, Dr. Tom Coon, Dr. Chris Goddard, Dr. Kay Holekamp, and Dr. Mike Sieflm .2 m $522 .35 u_:< - SEER 333.com. NOON ..n 3 8032 m E human—om Bob :Bohm - 3:2» 953%. Sow. ._e .e 520 ”32 ._e. 8 meta? $2 2 832% .528 02:22 - e38. e52“. mg. 530 E r.— wczwz 5:292 - 6.83.8. 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A similar ontogenetic shift has been described in signal crayfish (Pacifastacus leniusculus), where only juveniles show an alarm response to predatory eel odorants (Stebbing, 2004). Studies of blue crab show that stage I and stage IV-V instars respond with opposite behaviors to the same visual and chemical cues (Diaz et al., 2001). It makes sense that small fishes would be most alerted by alarm odorants because they are at greatest risk of attack. Hunger also mediates the degree of antipredator response when fish are exposed to alarm odorants. The first study to evaluate the effect of hunger on responsiveness to alarm cues was conducted by Smith in 1981 when he found that Iowa darters (Etheostoma exile), after fasting for 12 h, responded to a mixture of food and alarm odorants with a feeding response, but darters responded to the same odorant with an alarm response when satiated. Similarly, alarm responses in fathead minnows and reticulate sculpins (Cottus perplexus) are abolished after 24 h of fasting (Brown, 1996). Hunger of experimental fish may explain contradictory results obtained in lab and field studies of alarm odorants (Magurran et al. 1996), where in the lab fish are often fed to satiation, but in the wild food resources may be limiting. Age, sex, and maturity. Studies of the sea lamprey clearly illustrate how chemically-mediated behaviors are dependent on the age, sex, and maturity of 61 experimental animals. During sea lamprey migration, both males and females are extremely sensitive and attracted to migratory pheromones released by larvae (Sorensen & Vrieze, 2003), but sea lampreys become less responsive to migratory pheromones when the migration is complete and they begin to mature sexually. When fully mature, lamprey do not respond to migratory pheromones (PZS and ACA) (Bj erselius et al., 2000), but at that time, spermiated males construct spawning nests and release a mating pheromone component (3kPZS) that is highly attractive only to ovulated females (Siefl(es et al., 2005). Therefore, different ages and sexes of sea lamprey release and respond to chemical cues throughout their life history. Similar sex and maturity effects have been reported in the goldfish, where females sequentially release mixtures of pheromones throughout the ovulation cycle to prime males and induce courtship behaviors. Details will not be considered here because goldfish pheromones have been reviewed elsewhere (Sorensen & Stacey, 2004; Stacey, 2003; Stacey et al., 2003; Kobayashi et al., 2002; Sorensen et al., 1998; Sorensen, 1992). Consideration should be taken when working with a species with alternative mating strategies because dominate and sneaker (jack) males may differ in pheromone release and response. For example, dominate male black gobies (Gobius niger) respond aggressively to the ejaculate of other dominate males, but not to the ejaculate of sneaker males, likely because sneaker male ejaculate is pheromonally inconspicuous (Locatello et al., 2002). Even though sneaker males of most species typically do not release pheromones, evidence suggests that sneaker males in most cases are able to detect and respond to conspecific pheromones. For example, Yambe et al. 2006 used “jack” masu 62 salmon (Oncorhynchus masou) as bioassay subjects to identify female mating pheromones because small jacks were easy to work with in the lab. Sexual maturity is linked to increases in sex steroid concentration in animals with associated mating systems. Sex steroids function to physiologically ready an animal for mating, including enabling mature animals to detect and respond to mating pheromones. Carolsfeld 1997b showed in Pacific herring (Clupea pallasii) that individuals with high sex steroid concentrations were most likely to respond to spawning pheromones. In masu salmon and rainbow trout, responsiveness to pheromones was linked to sex steroid concentration when immature male parr, which do not naturally respond to female priming and mating pheromones, did indeed respond when treated with methyltestosterone (Yambe et al., 2003; Yambe & Yamazaki, 2001). Similarly, injection of androgens into male Barilius bendelisis increased responsiveness to female mating pheromones by activating male olfactory receptors (Bhatt et al., 2002). Overall, very little is known about the mechanism by which sex steroids act on the central nervous system and olfactory organ to modify responsiveness to chemical cues, but work in this area has just begun in the sea lamprey and the mechanism likely involves the hypothalamic-pituitary-gonadal axis (Chung-Davidson et al., in prep). Given the importance of sexual maturity in responsiveness, it is critical that the reproductive state and, when possible, sex steroid concentrations of experimental animals be described in mating pheromone experiments. Some field studies reviewed would have been more instructive if maturity of experimental animals had been carefully considered. For example, Young et al. 2003 conducted a field experiment to determine whether traps baited with mature male and female brook trout (Salvelinusfontinalis) captured a higher 63 proportion of animals than unbaited traps. Indeed, they found that traps baited with mature males captured more mature males than unbaited traps or traps baited with females. However, their in-stream experiments were conducted before most brook trout had spawned (inappropriate physiological context) and in a stream section containing no suitable spawning habitat (inappropriate environmental context). Their results would have been more informative and likely more convincing if their experiments had been conducted during the mating season on spawning riffles. Temporal variation in responses to chemical cues. Even if experimental animals are of the correct age, sex, and maturity, they may only respond to chemical cues at certain times of the day. Again, this point is clearly illustrated in the sea lamprey where migratory adults are nocturnal and only respond to migratory pheromones at night (Bjerselius et al., 2000). However, sexually mature sea lampreys are arrhythmic (Applegate, 1950), spawning night and day, and accordingly mature females respond to the mating pheromone night and day (Johnson et al., 2005). In the goldfish female priming pheromone, 17a, 20B-dihydroxy-4-pregnen-3-one (17,20, BP), robustly induced sexual arousal in males in the early morning when females are likely to be releasing 17,20, BP. However, males exposed to 17,20, BP in the afternoon showed lower levels of arousal (Defraipont & Sorensen, 1993). When planning behavioral studies, the life history of the experimental species should be carefully considered so that trials can be conducted at times when the species naturally responds to the chemical cue. Stress. The level of stress experienced by experimental animals should always be of concern in any animal behavior research. In most species, stress decreases responsiveness because increased cortisol concentrations lower sex steroids, which act to 64 reduce behavioral responses. Transport of animals from the field to the lab may be particularly stressful, and this presents a potential problem for lab experiments in which animals are obtained from the wild. This has been demonstrated in bull frogs (Rana catesbiana) (Licht, 1983), painted turtles (Chrysemys picta) (Licht, 1985), brown trout (Salmo trutta) (Pickering, 1987), and rainbow trout (Scott et al., 1994; Pottinger, 1992). Interestingly, Pacific herring respond more robustly to spawning pheromones when held in aquaria with shallow water (shallow water stressor). Spontaneous spawning may even occur when water is being drained from the tank (Carolsfeld et al., 1997b). Pacific herring naturally spawn in shallow turbulent rock reefs, so spontaneous spawning may be a natural response to a natural physical context. Regardless of whether stress increases or decreases responsiveness to chemical cues, the stress levels of experimental animals should be controlled so they are similar to those naturally encountered under field conditions. Social context, experience, and learning. In addition to physical and physiological contexts, social environment is an important context that influences responses to chemical cues (Ferrari et al., 2005). In goldfish, the effects of social context on male response to female priming pheromones 17,20 B-P and prostaglandin F 2a (PGF) have been investigated. When in social isolation, the male endocrine system is only primed by exposure to 17,20 [3P (Fraser, 2002). But the male endocrine system is primed by PGF when males are able to interact socially with females (Sorensen, 1989). In crustaceans, social context influences the release of mating pheromones and odorants in reproductive males (Breithaupt & Eger, 2002). 65 Female Chinese mitten crabs (Eriocheir sinensis) only release mating pheromones after physical interaction with a male (Herborg et al., 2006). Experience and learning by experimental animals has also been shown to alter behavioral responses of fish to alarm odorants. Several fish species have been observed to exhibit learned predator recognition to alarm odorants. Simultaneous exposure of an individual to a novel odorant and a stressful event, like chasing with a net, causes the individual to associate the odorant with danger (Kelley & Magurran, 2003; Chivers et al., 1995). Hatchery reared fish have been trained to recognize odors of natural predators, but the efficacy of this technique to reduce mortality of stocked fish has yet to be demonstrated (Mirza & Chivers, 2000; Brown & Smith, 1998). Zebrafish (Brachydanio rerio) have even been trained to associate red light with predation threat by simultaneously exposing them to alarm odorants and red light (Hall & Suboski, 1995b; Hall & Suboski, 1995a). Recognition of odorants can last up to a month, and such recognition has been demonstrated in field contexts (Pollock et al., 2003). Given these examples, the previous experiences of experimental animals should be carefully controlled, and test subjects should not be used more than once in any experiment. My literature search revealed that no chemical cue induces the same response in all individuals in all contexts. Responses to chemical cues are dependent on specific physical, physiological, and social contexts, all of which are interrelated and dependent on each other (Mikheev et al., 2006; Diaz et al., 2003; Hassler & Brockmann, 2001). If experiments cannot be conducted in the field, the most important contextual mediators of chemically-induced behaviors should be replicated and reported in the literature. The 66 best option would be to conduct experiments at the time and location in nature where chemically-mediated behaviors occur. 67 CONTRAINTS OF EXPERIMENTS IN FIELD ENVIRONMENTS Given how important it is to conduct chemical ecology experiments in natural contexts, one would expected that most research would be conducted in the field and would yield ecologically relevant results. But this is not the case because field research is constrained by the same physical and physiological contexts that are needed to elicit chemically-mediated behaviors. In the next section, major experimental and environmental factors that constrain field research will be discussed by reviewing field studies that yielded meaningful results despite the constraints imposed by nature. Experimental constraints. Observation of animals. One of the greatest constrains of experiments conducted in aquatic environments is the inability to observe behaviors because water is often turbid, deep, fast flowing, and structurally complex. Even if behaviors can be observed, care must be taken that observation techniques are noninvasive and do not alter animal behavior. In clear lakes or streams visual observation techniques have been used to record responses of fish to chemical cues (Johnson et al., 2006; Johnson et al., 2005; Johnsen, 1980). For example, the upstream movements of sea lamprey orienting toward mating pheromones have been documented by tracking individuals with radio telemetry and recording their movements on stream maps (Figure 3-2) (Johnson et al., 2006). Disadvantages of this technique are that data cannot be reviewed, some animals may be lost, and it requires a great deal of manpower. For example, during in-stream experiments two technicians were required to track one lamprey, and even then only half 68 Figure 3-2. Photograph illustrating two technicians manually tracking an ovulated female sea lamprey as it moves upstream towards a trap baited with spermiated male washings. One technician uses telemetry to locate the lamprey while the other technician records observed behaviors. This technique is labor intensive, requiring two technicians to track a lamprey. Image is presented in color. 69 of the lampreys that moved upstream to the pheromone were observed (Johnson et al., 2006). Other studies have used brief focal observations of individuals to obtain behavioral data (Siefkes et al., 2005; Liley et al., 1993), but observation times were short and behavioral data were lost. Perhaps the most adventurous observation technique employed to date has been snorkel surveys, in which fish were manually observed by divers. Snorkeling techniques have yielded several informative behavioral studies concerning alarm odorants and parental care in fishes (Golub et al., 2005; Neff, 2003), but require much manpower, and divers may disturb natural fish behavior. To avoid the drawbacks of directly observing behaviors, video cameras have been exploited to record behaviors in experimental sites spanning less than 10 m. Cameras, however, are of little use if animals need to be tracked over long distances and are especially susceptible to malfunction when used in harsh weather. For example, F inelli et al. 2000 monitored blue crab foraging behavior in a small tidal creek with a camera mounted above the water, but 206 of 323 observations had to be discarded because of poor quality. In many instances, pseudo-measures of behavior are collected when it is not feasible to continuously observe animal behaviors. This is especially true in studies of alarm odorants in fishes, where numbers of fish (or lack of fish) captured in traps baited with alarm odorants are taken as a pseudo-measure of alarm response (Pollock et al., 2005; Wisenden et al., 2004a; Pollock et al., 2003; Mirza & Chivers, 2001; Chivers et al., 1995; Mathis et al., 1995; Wisenden et al., 1994; Mathis & Smith, 1992). Another example is described in Wagner et al. 2006 in which sea lampreys were tagged with passive integrated transponders (PIT tags and antennas, Oregon RF ID) to determine when 70 they moved past defined stream locations while orienting towards migratory pheromones (Wagner et al., 2006). PIT tagging systems are a pseudo-measure of behavior because they only inform us about when lampreys passed a particular stream location and do not record the speed, direction of movement, or other pheromone-induced behaviors. It is essential that pseudo-measures of behavior correlate with the chemically-mediated behavior of interest in order to draw informed conclusions. With determination, creativity, and new technology, researchers can overcome obstacles of observing animals in field studies and can adequately describe chemically-mediated behaviors. Experimental animals. When designing field experiments it is important to consider the source and history of experimental animals. Most field studies use animals naturally present in the environment as test subjects (Olsen et al., 2006; Carton & Montgomery, 2003; Brown et al., 2001b; Finelli et al., 2000; Zimmerfaust et al., 1995). This approach ensures that responses to chemical cues are relevant in natural populations. However, using wild animals limits experimental design because it is difficult to identify individuals, obtain physiological data on test subjects (size, age, sex, maturity), evaluate social interactions, control for past experiences, ensure sample size will be adequate (Scholz et al., 2000), and determine whether the same animal was tested more than once (pseudo-replication). Some studies attempt to avoid pseudo-replication by making experimental sites hundreds of meters apart (Brown et al., 2001b). Other researchers, like Zimmer et al. 1999, evaluated the likelihood of pseudo-replication by estimating the density of the experimental species in the field site using mark-recapture techniques. To avoid the constraints of experimenting on animals naturally present in the environment, sea lampreys have been released into experimental streams during 71 experiments. To do this, wild lampreys were captured in traps and allowed to naturally mature in holding cages (1 m3) located in spawning streams. Once fully mature, females were tagged and then released into the experimental stream segment (Johnson et al., 2006; Wagner et al., 2006; Johnson et al., 2005; Siefl6 803 Saw 8535me .35 Bannmmem 25 2:38 8353: m:m>oE 0:53 03:55 @8255 some 3 :81: 55896:: 835356 35.3 :88 535855 5:: $558022: 835.0530: .50 .5565: 33.5 :88 .Emo5m:3oQ 53: 50 85:5: 26$ :88 53% ”:85 85:0 5:: owmu ammo—8 25 :85 :53m 8 0:55 :23 :38 USE. ”3:5 “:58meme :_ 35:98 803 5:5 mac—«Eek 58:35 Mo 52353 8552563 558550 .835 25 2 tom—nae 33 mmem 553 3:5 3:3-mmem E 583:8 203 33:55 3:295 =< A3558 5:028 65:8 :53 52:3 803 £55 505 5:3 5:: .mNa—v—m :53 3:8 33 95 .550 05 5:: 5:038 65:8 553 3:3 33 95 0:0 :23 35:98 203 5:: 585mm: BEE :55 3385 3:230 50 85:5: 05 fl :05:£5m5 .385 SE 312:5 585: 50830:: 835mm: Bangsmch—m «.-— 935,—. 104 (binomial distribution, p = 0.012). Behavioral observations show that ovulated females oriented towards 3kPZS- baited traps by swimming directly upstream (Figure 1-3). Ovulated females captured in traps baited with 3kPZS at 10‘1 ‘, 10'”, or 10'13 M did not differ in time taken to swim upstream into the trap after leaving the release cage (mean = 18.1 min, range 2.7 to 84.8 min), the number of rests (mean = 3.8, range 0 to 16), number of downstream movements (mean = 0.2, range = 0 to 2), or number of sidestream movements (mean = 1.3, range = 0 to 7) (Table 1-3). Only 17% of ovulated females exhibited two or more sidestream movements while moving upstream toward the 3kPZS-baited trap. Ovulated females located the exact release point of 3kPZS even when concentrations varied 1000 fold. I further reasoned that ovulated females would not become adapted to 3kPZS even after prolonged exposure in 3kPZS plumes during their directed upstream movement over long distances and in diverse river habitats. This hypothesis was tested by recording ovulated female responses to 3kPZS over a 650 m distance at two experimental sites in the Ocqueoc River. One segment was located on the sea lamprey spawning riffle used in previous experiments and the other site was located several km downstream of the spawning riffle which was characterized by slow deep flow and sandy bottom (run) (Figure 82). At each experimental site, ovulated females were released 650 m downstream of a trap baited with 3kPZS to reach 10'12 M and a trap baited with control solvent. 3kPZS induced directed upstream movement over 650 m in both environments. In the riffle and run stream segments, the proportion of ovulated females moving upstream and entering within 1 m of 3kPZS-baited traps did not differ, showing that 105 Figure 1-3 3kPZS-baited traps capture all females when compared to unbaited traps. Observed movements of individual ovulated females trapped when 3kPZS was applied at 10'11 M, 10‘12 M, or 10‘13 M in a randomly selected trap and when control solvent was applied in the other (Trap, and Trapk). Red lines illustrate ovulated females entering the left trap when the lefl trap was baited with 3kPZS. White lines illustrate ovulated females entering the right trap when the right trap was baited with 3kPZS. Green illustrates ground and black illustrates river. Figure displayed in color. 106 3kPZS induced equally strong migrations in both habitats (Fisher’s exact; p = 0.738). More ovulated females moved upstream and entered within 1 m of the baited traps when 3kPZS was applied to the stream than when control solvent was applied to both traps (Table 81 and herein; Fisher’s exact; riffle: p < 0.001; run: p = 0.005). However, the proportion of ovulated females captured in 3kPZS-baited traps was greater in the riffle habitat than in the run habitat (Fisher’s exact; p = 0.002) and the time for ovulated females to enter within 1 m of the 3kPZS-baited trap was longer in the riffle stream segment than the run segment (Wilcoxon rank-sum test; p = 0.027, 2 = -2.21, df = 30). Slower swimming speeds of ovulated females in the riffle may be due to fast water velocity and the inefficiency of anguilliforrn swimming. Similarly, high water velocity through the trap at the riffie site may have caused ovulated females to swim with great effort into the trap funnel resulting in higher capture efficiency. 3kPZS elicits long upstream migration in both environments, but the trapping techniques used in this study may be most efficient if traps are placed in riffle environments. Role of 3kPZS as a component of the pheromone mixture. Given that most characterized pheromones are mixtures which only elicit strong responses when all components are present (17), it was interesting to observe that 3kPZS alone induced robust upstream movements over long distances and ranges of concentrations. It has been hypothesized that spermiated male washings (SMW) contain additional pheromone components (18) that induce near source search behaviors in ovulated females (7). Thus, I wished to confirm the role of 3kPZS—mediated upstream movement when placed in the context of SMW by directly comparing responses of 107 ovulated females to synthesized 3kPZS and SMW over long and short distances. SMW were used instead of live spermiated males to provide an unequivocal test of whether additional pheromone components induce near source search behaviors. Previously, it was found that behavioral responses of ovulated females to spermiated males or their washings in a two-choice maze do not differ (7). In a natural stream, traps baited with spermiated males and SMW both capture large proportions of ovulated females (16, 19). These results are not surprising since ovulated females are blind (15) and naris-plugged ovulated females are not able to locate spermiated males over long or short distances (19). Therefore, by comparing 3kPZS and SMW, I also evaluated the potential utility of 3kPZS in redirecting ovulated females away from natural sources of pheromone, and thus a potential mate. At the spawning riffle segment, a lamprey nest was constructed in each river channel 45 m upstream of the confluence of the channels (Figure 83a). In one nest SMW was applied to reach an in-stream natural 3kPZS concentration of 7.5 X 10'13 M. In the other nest (other channel) synthesized 3kPZS was applied at 0.7, 1.0, 1.3 or 3.3 times the concentration of natural 3kPZS in SMW. Females were released 250 m downstream and had to choose which channel to enter 45 m downstream of the odor sources. Surprisingly, when applied at equal 3kPZS concentrations, synthesized 3kPZS and SMW attracted equal proportions of ovulated females, and at merely 3.3 times the concentration of 3kPZS in SMW, synthesized 3kPZS attracted 84% of responsive ovulated females (Table 2.3). Notably, nest observations show that ovulated females spent 10 fold more time in nests baited with SMW than nests baited with 3kPZS (Table 2-3). 108 a; 55 :3 we 2 a: 5 fig : a a 32m .9 3%: 2 2-2 ”e a. so: :85 32. 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To confirm this finding, the near source effects of 3kPZS and SMW were compared by building two spawning nests 1.25 m apart (Figure S3b) and applying SMW to one nest to reach a natural 3kPZS concentration of 7.5 X 10'13 M and synthesized 3kPZS to the other at 1.3 or 3.3 times the concentration of natural 3kPZS in SMW. Contrary to results from 45 m comparison experiments, all ovulated females went to the SMW when synthesized 3kPZS was applied at 1.3 times, and equal proportions of ovulated females visited both nests when 3kPZS was applied at 3.3 times (Table 2-3). Again, SMW retained ovulated females about 10 times longer than synthesized 3kPZS. Why did SMW and 3kPZS equally attract ovulated females to nests over a 45 m distance when applied at 7.5 X 10'13 M, but synthesized 3kPZS did not retain females on nests? It is possible that, at this 3kPZS concentration, the pheromone components within SMW that attract and retain ovulated females near nests may not be detected long distances downstream due to lower release rates or olfactory sensitivities (or both). To investigate this possible scenario, 3kPZS and SMW were directly compared at a 45 m distance across a 1000 fold change in concentration. Responses to SMW and 3kPZS were directly compared when the 3kPZS concentration of both sources were equal to 10’ ”, 10'”, 10'”, and 10"4 M, respectively. At each concentration, 3kPZS and SMW triggered equal proportions of ovulated females to move upstream into the baited channels (Table 2-3 and herein), showing that within the range of concentrations tested, 110 3kPZS is the only pheromone component that influences long distance responses in ovulated females. As expected, retention in the SMW nest was significantly higher than 3kPZS at 10'11 M and 10''2 M. However, retention in the SMW nest and 3kPZS nest did not differ at 10''3 M and 10''4 M, perhaps because at extremely low concentrations minor components were not detectable even when ovulated females were on the nest. 3kPZS disrupts female orientation to male pheromone. Given the dominant role 3kPZS plays in the pheromone mixture to induce upstream movement over various distances, I postulated that high concentrations of 3kPZS can disrupt both near and far source effects of the natural male pheromone blend. An experiment was conducted to test this hypothesis and further confirm that 3kPZS indeed maintains robust movement directly upstream over a wide range of concentrations. At the spawning riffle, SMW was applied to reach an in-stream natural 3kPZS concentration of 10'12 M 20 m upstream of the ovulated female release site and a background synthesized 3kPZS source was applied 40 m upstream of the SMW source, so that the synthesized 3kPZS-plume enshrouded the SMW-plume (Figure 2—3). Background concentrations of 3kPZS applied were 0 (vehicle solution), 10'”, 10'l l, or 10'‘0 M. Consistent with the hypothesis, 3 higher proportion of ovulated females completely missed the enshrouded SMW source while swimming upstream to the 10'10 M synthesized 3kPZS source than to the control source (Table 3-3 and herein; logistic regression; X2 = 4.24, df = 1, p = 0.040). To discern the mechanism for this disruptive effect, individual ovulated female movement tracks were compared to the plume 111 Figure 2-3 Female movement tracks during 3kPZS disruption experiments. Synthesized 3kPZS was released 40 m upstream of a source of spermiated male washings (SMW) with natural 3kPZS at 10''2 M. Color scale illustrates estimated 3kPZS molar concentrations from both sources of 3kPZS throughout the stream. Background 3kPZS concentrations achieved when fully mixed with the stream discharge. (a) No 3kPZS background. (b) 3kPZS 10'12 M background. (0) 3sz3 10'“ M background. 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When 3kPZS 10''0 M was applied, ovulated females were more widely distributed across the stream and were less likely to track the highest concentration of natural 3kPZS originating from SMW (Figure 2-3; Table 82; Figure S4; ANOVA; F = 5.795, df = 3/20, p-value = 0.005). Furthermore, a higher proportion of ovulated females swam upstream of the SMW source when 3kPZS was applied at 10'”, 10'l ', or 10''0 M than when control solvent was applied. For those ovulated females that did visit the SMW source, they spent less time within 0.5 m of the SMW release point when 10'll M or 10'lo M synthesized 3kPZS was applied upstream (Student’s t-test; t—value = -2.81, DF = 81, p = 0.063 and t-value = -2.61, DF = 81, p = 0.011, respectively). When ovulated females moved upstream of the background source of synthesized 3kPZS they exhibited more sidestream and downstream movements (Table S3). These experiments also confirm that ovulated females display robust upstream movements to 3kPZS over concentrations ranging lOO fold. First, the proportion of females that moved upstream and located a source of SMW or 3kPZS did not differ when 3kPZS concentration varied from 10'12 M to 10''0 M (Table 3-3). Second, swimming speed and swimming distance (Table S4) from SMW to the 3kPZS source did not differ among 3kPZS concentrations. Third, time spent within 0.5 m of the 3kPZS source did not differ among 3kPZS concentrations (Table 3-3). 114 DISCUSSION In natural spawning streams, synthesized 3kPZS applied over a wide range of concentrations lured ovulated females to swim upstream over long distances and subsequently enter traps. Efficient localization of potential mates is essential for sea lamprey to bring their complex life history to fruition in a single spawning event over a few days before senescence (15). The male sea lamprey mating pheromone facilitates mate finding by signaling to ovulated females the location of spawning grounds and individual nests. 3kPZS-mediated upstream movement is sufficient and efficient in directing ovulated females to individual nests. A single source of 3kPZS triggered the same directed response in ovulated females over distances of 70 m and 650 m and concentrations varying from 10''0 M to 10''4 M. It is adaptive for ovulated females to display 3kPZS-induced upstream movement over highly diverse conditions because flow and male abundance differ greatly within and among spawning streams, causing 3kPZS plumes to vary greatly in intensity, and in temporal and spatial profiles. Ovulated females appear to employ the simple orientation strategy of swimming upstream when 3kPZS is detected and moving back downstream and casting side-to-side when the signal is lost. Not only did a vertebrate swim up pheromone plumes, but its efficacy in locating the pheromone sources is comparable to those known in insects. This can be attributed in part to the predictability of shallow river pheromone plumes, which are essentially confined in a one dimensional space by the width, depth, and unidirectional flow of water 115 (20). Moths in a forest enviromnent orient to unpredictable airborne pheromone plumes in a three dimensional space by moving upwind when the pheromone is detected and casting from side to side (optomotor amenotaxis) when the scent is lost (21). Unlike insects, ovulated females oriented toward a single source of 3kPZS by swimming directly into the unidirectional flow (Figure 1-3). In the disruption experiments, where an additional source of 3kPZS was located upstream of SMW, ovulated females that bypassed the SMW moved directly upstream to the background source of 3kPZS and subsequently showed more sidestream and downstream movements when ovulated females bypassed the background 3kPZS source (Figure 2-3). Ovulated females may use casting as a behavior to ensure that they do not overshoot the spawning grounds and bypass possible mates. It is notable that when ovulated females lose the 3kPZS signal, it takes several seconds (many sniffs) to begin casting, suggesting that either there is a significant integration time to recognize that the signal has been lost, or that upstream movement, once triggered by the pheromone, continues for a period governed by an internal mechanism, as proposed for moths (22). Sea lamprey and moths appear to use similar casting strategies, albeit on different temporal and spatial scales, to relocate lost plumes. A distinct difference is that in a one-dimensional environment, when the odor is briefly lost, it may not be advantageous to irrunediately move sidestream because plume intermittency may be related to distance from the source rather than location within the stream channel. I In many insects, sex pheromones, like most natural odors, are typically blends of components in specific proportions, with two or more being necessary to elicit a behavioral response (10). In particular, robust long distance behavioral responses are 116 sometimes only elicited when a blend of compounds which function as one signal are present (17). 3kPZS alone elicited robust upstream movements over long distances and was equally effective as the whole pheromone blend found in SMW from 10"1 M to 10'14 M at attracting ovulated females at a 45 m distance (Table 3-3). However, males may excrete additional components which function over short distances to retain ovulated females on the nest. In experiments directly comparing 3kPZS and SMW released into nests separated by 1.25 m, SMW retained ovulated females 10 fold more time than 3kPZS (Table 2-3). Additional evidence that males release additional compounds is gleaned from disruption experiments when 3kPZS 10"0 M was applied 60% of responding ovulated females located and were retained at source of SMW even when background synthesized 3kPZS l m downstream of the SMW source was 5 times greater than the 3kPZS present in the SMW as determined by dye tests (Figure 2-3). Recently, several compounds have been isolated from larval sea lamprey washings and subsequently shown to modify behaviors of migratory adults in two-choice mazes (23). Collectively, results show that 3kPZS is a component of the pheromone that functions independently to elicit long distance upstream movements in ovulated females, directing them to nests, and that unidentified components induce near source attraction and retention. The mechanism by which the male sea lamprey mating pheromone coordinates mate finding and reproduction closely resemble the “component” mechanism first described in the pine beauty moth (Panolisflammea) (24) and recently described in the red-legged salamander (Plethodon shemani) (25) where each component of the pheromone induces separate behaviors such as attraction, landing, or copulation (24). The data collected in this study do not support the “blend” hypothesis (17), in which all 117 pheromone components work as one signal to induce all behaviors. However, these hypotheses should be re-evaluated when all pheromone components are identified. From an applied standpoint, the data show that a synthesized pheromone modifies the behavior of ovulated females in their natural habitat and demonstrate the possible utility of 3kPZS as the first synthesized vertebrate pheromone control agent. This hypothesis should be further tested by comparing the effectiveness of 3kPZS versus spermiated males in their natural habitat. Capture rates and effective distances of 3kPZS- baited traps were similar to or greater than those reported in insects (14); but unlike mixtures of insect pheromones that attract males, 3kPZS alone induced robust responses in females. These are distinct advantages of 3kPZS because removal of ovulated females will result in a proportional reduction in viable eggs, and thus be more effective than removal of males. A single compound is less expensive to synthesize, easier to apply, and requires less testing to register with regulatory agencies. In addition to trapping, 3kPZS may be used to divert ovulated females away from natural male pheromones or redistribute ovulated females to tributaries not suitable for spawning or survival of offspring. This approach may be highly effective because sea lamprey only use about 6% of streams in the Great Lakes basin to spawn (5), and in any particular stream sea lamprey use a small portion of habitat for spawning (15). Furthermore, an “all or nothing” response to 3kPZS over a wide range of concentrations makes control applications even more efficacious because high concentrations are not required to induce strong responses. In the end, 3kPZS-based techniques may provide an environmentally benign means of managing sea lampreys in the Laurentian Great Lakes, where only 270 g of 3kPZS would be required to activate all currently trapped streams in Lakes Huron, 118 Michigan, and Superior at 10‘'3 M during the 3 week spawning period (5.5 trillion liters of water). 119 MATERIALS AND METHODS Behavior tests and permit. Use of sea lampreys was approved under Michigan State University Institutional Animal Use and Care Committee permit 05/06-066-00. Application of 3kPZS and related bioactive components was approved by the Michigan Department of Environmental Quality and United States Environmental Protection Agency through experimental user permit 7543 7-EUP-2. Experiments were conducted in the Ocqueoc River, MI, USA (7, 16), in stream segments historical infested with larval and spawning- phase sea lampreys (15), however a barrier several km downstream currently prevents sea lamprey infestation. 3kPZS concentrations were calculated as the final in-stream concentration when completely mixed with the whole stream discharge. Dye tests confirmed that 3kPZS was thoroughly mixed 70 m downstream of the application point. 3kPZS was custom synthesized by Bridge Organics (Vicksburg, Michigan, USA) at purity higher than 95%. A single batch of SMW with a natural 3kPZS concentration of 1.85 mg/L was used in all 3kPZS verse SMW direct comparison experiments in 2007 and a single batch of SMW equaling 3.27 mg/L was used in 2008 direct comparison experiments. A single batch of SMW with natural 3kPZS concentration of 1.5 mg/L was used in all 3kPZS disruption experiments. Ovulated females were fitted with external radio tags (Model 393, Advanced Telemetry System, Isanti, Minnesota, USA) and tracked using direction radio antenna and receiver (Lotek Engineering Incorporated, Newmarket, Ontario, Canada) (7, 16) during 70 m trapping experiments. In all other experiments, ovulated females were fitted with external passive integrated transponders 120 (PIT tags) and tracked with PIT tag antennas connected to a multiplexer (Oregon RFID, Portland, Oregon, USA). Females were released in groups of three to five for 70 m trapping and in groups of six to 11 for all other experiments. Visual observations of random ovulated females were recorded on stream maps using stream markers as reference points (1 9). Behavioral Statistics. Female behaviors were assumed to be independent as observed from earlier studies (7, 16). Binary data from experiments with more than two treatment groups were evaluated with logistic regression and models showed no evidence of overdispersion or nonlinearities. Binary data from experiments with two treatment groups were evaluated with a nonparametric Fisher’s Exact Test. Time variables and orientation behaviors were evaluated with general linear models where time variables were square-root transformed and orientation behaviors were square-root transformed or In transformed when needed to meet model assumptions of residual heteroscedasticity and normality. Time data in 650 m trapping experiments were evaluated with a nonparametric Wilcoxon rank-sum test because data could not be transformed to meet parametric statistic assumptions. For 3kPZS verse SWM direct comparison and disruption experiments, data were also analyzed with mixed effect logistic regression and mixed-effect general linear models with a random effect of trial date. All statistical results from general linear models are robust to the inclusion of the random effect of trial date, supporting the assumption that a single ovulated female can be treated as an individual sample (7). Statistical results reported are from two-tailed analyses. A listing of the statistical tests and transformations conducted are in Table SS. 121 ACKNOWLEDGEMENTS I thank the staffs of US. Geological Survey Hammond Bay Biological Station and US. Fish and Wildlife Service Marquette Biological Station for facilities, sea lamprey, and equipment. The Staff of US. Geological Survey Upper Midwest Sciences Center helped obtain pheromone use permits from U. S. Environmental Protection Agency. Dr. John Teeter and Dr. Michael Siefl96.8 for 3kPZS and m/z 476.3>97.8 for 3kPZS-d5. Calibration curve was established between 10 pg - 2 ng/injection. Trap Design. Traps identical to those described in Johnson et al. 2006(3) were used, with the following modifications. Two 1.1 m plastic mesh (1 cm in diameter) leads were extended from the downstream funnel at 450 angles. Two sandbags (0.60 m x 0.25 m x 0.25 m) were placed on the upstream side of each lead to deflect water away from the trap and one sandbag was placed in the upstream funnel to slow the velocity of water flowing through the trap (Figure 81 and 82). No block net was placed upstream of the trap. These trap modifications may have aided capture efficiency, but the study did not test the utility of the modifications. Detailsfor specific behavior experiments. 134 Female orientation to 3kPZS-baited traps from 70 m. Side of 3kPZS treatment was randomly determined by flipping a coin for each trial. 3kPZS treatments and control treatment were randomized without replacement by drawing numbers out of a hat. Ovulated females were pre-exposed to the odorants for 30 min prior to release. Five trials were conducted for each treatment from 10-June-2005 to 1-July-2005 between 0700 and 1500. Female orientation to 3kPZS-batted traps from 650 m in riffle and run stream segments: Stream segment descriptions. Stream segments were in the Ocqueoc River, MI, USA. The riffle stream segment was located on a historic sea lamprey spawning riffle (Township 35N, Range 3E, Sections 27 and 34) and was the primary site used throughout this study since it is the natural habitat in which sea lamprey spawn. The riffle segment is characterized by water velocities around 0.60 m/sec, depths between 0.1 and 0.5 m, and a substrate nearly 100% composed of rubble and coarse gravel (Figure S 1 ). The run stream segment was located approximately 10 km downstream of the riffle stream segment (Township 36N, Range 3E, Section 20). At this site, the river is characterized by water velocities around 0.15 m/sec, depths between 0.5 and 1.0 m, and a substrate nearly 100% composed of sand and clay (Figure 82). A more detailed description of the two sites can be found in Applegate 1950 (4). Female orientation to 3kPZS-batied traps from 650 m in riffle and run stream segments: Experimental design. At both experimental sites a mixture of 3kPZS 10"2 M and 701, 1201-dihydroxy-5a-cholan-3-one-24-oic acid (3-keto allocholic acid; 3kACA) 10' '3 M was applied to the pheromone-baited trap. The active component applied to the traps was 3kPZS 10’'2 M because all experiments conducted to date show that 3kACA 135 does not elicit behaviors in ovulated females (Chapter 5). Side of 3kPZS treatment was randomly determined by flipping a coin. Ovulated females were pre-exposed to odorants for 1 h and released for 8 h. Movements into the trapping area were determined with an across channel PIT antenna arrayed 30 m downstream of the traps. Movements near the traps were determined with 2 m2 PIT antennas arrayed around the outside perimeter of the traps to determine if ovulated females encountered the trap (entered within 1 m of odor source). Traps were checked at the end of the experiment for ovulated females. At the riffle stream section, four 3kPZS treatment trials and three control trials were conducted from 2-June-2006 to 10-June-2006 between 0600 and 1800. At the run stream section, six 3kPZS treatment trials and 2 control trials were conducted from l-August- 2005 to 8-August-2005 between 0700 and 1800. Female preference for 3 kPZS and spermiated male washings at 45m downstream. Sandbags were used to prevent water from mixing between channels through the island and sandbags were used to extend the island 12 m further downstream (Figure S3). It was confirmed that no surface water was mixing between channels by conducting rhodamine dye tests and analyzing water samples with a fluorometer. Across channel PIT antennas were arrayed at the downstream end of each channel to record movements into each channel. Pheromone was pumped into the middle of a man-made sea lamprey spawning nest approximately 0.5 m in diameter, which is a typical size of a lamprey nest in the Ocqueoc River (4). Square PIT antennas (1 m2) were placed around each nest to record entry and retention within 0.5 m of each odorant. In the first set of comparisons testing SMW with 3kPZS equal to 7.5 x 10''3 M, females were pre-exposed to the odor for l h and released for 3 h. Test odorants were 136 switched between channels for each new trial. The four synthesized 3kPZS treatments were conducted at different times and dates: 3kPZS 5 X 10'l3 M (0.67 times) was tested l2-June-07 to 15-June-07 between 0800 and 1200; 3szs 7.5 x 10'13 M (1.0 times) was tested 24-July-07 to 27-July-07 between 1900 and 0300; 3szs 10'” M (1.33 times) was tested 7-July-07 to l3-July-07 between 1830 and 0200; and 3kPZS 2.5 X 10''2 M (3.3 times) was tested 5-July-07 to 12-July-07 between 1830 and 0100. Four trials were conducted for each 3kPZS treatment. Differences in female retention within 0.5 m of pheromone sources were evaluated with general linear models where retention was explained by the pheromone visited. In the 3.3 times 3kPZS verse SMW experiment, the nest effect was included in the model because it significantly improved model fit (Likelihood ratio test p = 0.009; DF =1/l7; F-Stat = 8.78); meaning that in this experiment, ovulated female retention times were different between nests, regardless of pheromone treatment. Direct comparisons of 3kPZS and SMW at 10'1 l, 10'”, 10'”, and 10'14 M were conducted between l4-July-08 and 7-Aug—08 between 2000 h and 0230 h. Four trials were conducted at 10''2 M, six trials were conducted at 10'” M and 10'13 M, and 10 trials were conducted at 10'M M. The same test system was used as described above, with the exception that ovulated females were only released for 2 h after a 1 h odor exposure period. During 2008 experiments, ovulated females showed a significant preference for the right channel regardless of pheromone treatment (10'll M: p = 0.002, df = 1, X2 = 9.17; 10*” M: p<0.001, df= 1,,r2 = 22.36; 10"3 M: p<0.001, df= 1,)(2 = 16.43; 10''4 M: p = 0.001, df = 1,X2 = 6.22). To confirm that females would enter the left channel if a more preferred odorant was applied, the left channel was baited with synthesized 3kPZS 137 at 2 x 10''2 M and the right channel was baited with 3sz3 at 10''2 M. Three trials were conducted from 26-July-08 to 28-July-08. Fourteen of 16 responding females entered the left channel baited with 3kPZS 2 x 10'12 M, which is significantly greater than the proportion expected to enter the left channel given a 50/50 binomial function (p = 0.002). Female preference for 3 kPZS and spermiated male washings when separated by 1.25 m. Four trials were conducted for each treatment from 28-July-O7 to 3 l -July-07 between 1900 and 0430. Test odorants were switched between nests for each new trial. Cross channel PIT antennas were arrayed at the downstream end of each channel and square antennas (1 m2) were placed around each odorant. Females were pre-exposed to the odor for 1 h and released for 3 h. 3kPZS disruption experiments. 3kPZS treatments were randomized without replacement by drawing numbers out of a hat. PIT antennas were arrayed across the stream channel 20 m and 60 m upstream of the ovulated female release point and square antennas (1 m2) were placed around each odorant. Four trials were conduced for each treatment from 30-June-2006 to l7-July—2006 between 0700 and 1300. To quantify ovulated female orientation in relation to estimated 3kPZS concentrations, three grid systems, aligned parallel to streamflow, were overlaid on maps illustrating lamprey movements. The lower grid covered 20 m downstream to 0 m downstream of the SMW release point; the middle grid covered 0 m upstream to 20 m upstream of SMW; the upper grid covered 20 m downstream to 0 m downstream of 3kPZS release point. The size of a square in the grid was approximately 1 m2. Average dye concentration within each square was calculated. Squares within each row were ranked according to average dye concentration within that block; where 1 rank indicated 138 the highest average dye concentration within that row. In the lower grid, blocks were ranked according to the average concentration of dye originating from the SMW release point. Using the grid ranking system, ovulated female distribution in the stream was evaluated by numerating the number of times ovulated females entered blocks of given rank. Then the percentage of times ovulated females entered a block of a given rank was calculated by dividing the number of times ovulated females entered a block of a certain rank by the total number of blocks that ovulated females entered. Differences in the percentage of times ovulated females entered a block of a given rank among 3kPZS treatments were evaluated within each ranking grid system using a general linear model where the percent times ovulated females entered a block was explained by the rank of the block, the treatment, and the interaction between rank and treatment. The distance each lamprey swam in each grid was determined by using an opisometer (ATM, Swiss) to measure the length of each lamprey track on the raw data sheets. Map distances were converted to stream distances using the map scale. Differences in average distance ovulated females swam among 3kPZS background treatments within each grid system were evaluated with a general linear model where swimming distance was explained by treatment. Dye tests. 3kPZS disruption dye tests. During 3kPZS disruption experiments, dye tests using rhodamine (Turner Designs, Rhodamine WT, Sunnyvale, CA, USA) were conducted to model 3kPZS dilution and distribution in the stream. In the first test, 139 rhodamine was applied at the SMW release point to reach a final in-stream concentration of 1.0 ug/L. In the second test, rhodamine was applied at both the SMW and 3kPZS release points to reach a final in-stream concentration of 1.0 ug/L. After a 10 min dye introduction period, water samples were collected in 5 ml glass vials in transects across the stream at every 0.5 m. Stream transects were located every 5 m when greater than 10 m downstream of a pheromone source and every 2.5 m when less than 10 m downstream of a pheromone source. The florescence intensity of each sample measured at 556 nm was determined in a luminescence spectrometer (Perkin Elmer LSSSS, Downers Grove. IL, USA) and rhodamine concentration was estimated using a standard curve (R2 = 0.9998). Because a test was not conducted when dye was only applied at the 3kPZS application location, the concentration of dye originating from the 3kPZS application location was estimated by calculating the difference in rhodamine concentration between the second and first dye tests at each sampling point. Rhodamine originating from the 3kPZS application location was increased by 10 and 100 times at each sampling point to estimate 3kPZS treatments of 10''1 M and 10'10 M, respectively. 3kPZS vs. SM W 45 m dye tests (Figure S3a). Three dye tests were conducted with rhodamine as described above: 1) Dye was applied to the pheromone release point in the left channel, 2) dye was applied to pheromone release point in the right channel, and 3) dye was applied to both pheromone release points. Dye was sampled in stream transects and analyzed as described above. Dye map programming. Dye concentration in the stream was modeled by a Monotonic Piecewise Cubic Hermite Interpolation Polynomial (5). Stream maps, dye distribution maps, and ovulated female movement tracks were produced in Python 140 (Version 2.4, http://wwwpythonorg/ Copyright © 1990-2006, Python Software Foundation) and Python Imaging Library (Version 1.1.6 http://www.pythonware.com/ products/pil/ Copyright © 1997-2006 by Secret Labs AB & Copyright © 1995-2006 by Fredrik Lundh, Publisher: Secret Labs AB) and exported to Photoshop (Version C82) for final display. Dye map color contours were exponentially scaled to match the back calculated concentration of 3kPZS in the stream. 141 SUPPORTING REFERENCES 1. Vladykov VD (1949) Quebec Lamprey. List of Species and their Economic Importance. (Province of Quebec Department of Fisheries, Quebec). 2. McMahon TE, Zale AV, Orth DJ (1996) in Fisheries Techniques Second Edition, eds Murphy BR, Willis DW (American Fisheries Society, Maryland), pp 83-120. 3. Johnson NS, Luehring MA, Siefkes MJ, Li W (2006) Pheromone induced behavior and pheromone reception in ovulating female sea lampreys. N. Am. J. Fish. Manage. 26:88-96. 4. Applegate VC (1950) Natural History of the Sea Lamprey in Michigan (U. S. Department of Interior Fish & Wildlife Service, Washington DO). 5. Fritsch FN, Carlson RE (1980) Monotone Piecewise Cubic Interpolation. SIAM J. Numer. Anal. 17:238-246. 142 CHAPTER 4 IN-STREAM RESPONSES OF PRE-OVULATORY FEMALE SEA LAMPREYS TO PUTATIVE MIGRATORY AND MATING PHEROMONE COMPONENTS 143 ABSTRACT On their journey to locate suitable spawning streams and habitat within streams, sexually immature adult sea lampreys (Petromyzon marinas) are directed by migratory pheromones excreted by larval lampreys. A mixture of petromyzonamine disulfate (PADS), petromyzosterol disulfate (PSDS), and petromyzonol sulfate (PZS) induce preference responses in migratory sea lampreys in a two—choice maze. When sexually mature, female sea lampreys are directed upstream to spawning nests by a mating pheromone component, 3-ketopetromyzonol sulfate (3kPZS), released by spermiated males. The objective of this study was to test the hypotheses that PADS, PSDS, and PZS direct in-stream migratory behavior of pre-ovulatory females, and that 3kPZS functions as a mating pheromone to direct upstream movement of only ovulated females. In-stream preference responses of pre-ovulatory females were recorded when exposed to three different groups of compounds: a mixture of synthesized PADS, PSDS, and PZS; a mixture of PADS, PSDS, PZS, and 3kPZS; and 3kPZS alone. 3kPZS induced directed upstream movement in pre-ovulatory females that did not differ from that of natural migratory pheromone over long distances. A mixture of PADS, PSDS, and PZS only induced directed upstream movement when spiked with 3kPZS. Results demonstrate that 3kPZS, not a mixture of PADS, PSDS, PZS, directs the upstream migration of pre- ovulatory females. 3kPZS is likely an aggregational pheromone that directs upstream movement of sexually mature and immature lampreys. These results have major implications for pheromone-based management of sea lamprey in the Great Lakes, where 144 it now appears that 3kPZS may be used to lure sea lampreys into traps during the migratory and spawning period. 145 INTRODUCTION Pheromones are used by numerous fishes to elicit specific, adaptive behaviors or physiological responses in conspecifics (Chapter 1). Behavioral responses of fishes to pheromones in lakes or streams have rarely been described because it is challenging to unobtrusively observe fishes (Chapter 2) and few pheromones have been chemically identified and synthesized (Brennan & Zufall, 2006). Thus, most fish pheromone research has been conducted in laboratory contexts. Due to increased complexity in contextual regulation of behavior (Stowers & Marton, 2005; Ziegler, 2005) and motor systems (Mason, 1989; Novotny et al., 1986), fishes may not be expected to always respond naturally to pheromones within the constructs of laboratory conditions (Chapter 2). Sea lamprey (Petromyzon marinus) pheromones have been extensively studied in laboratory contexts (Bjerselius et al., 2000; Adams et al., 1987; Teeter, 1980), but recently synthesized compounds have allowed research to be conducted in natural streams (Siefl 0.5, df=1,X~’ = 0.01; 158 Figure 2-4. Oocytes of a pre-ovulatory female taken from the batch of lampreys used for field experiments testing putative migratory and mating pheromones. Oocytes displayed were immature and did not have a nucleus or columnar follicular cells. About 80% of oocytes examined were in this stage of maturity. Y = yolk. Figure presented in color. 159 Figure 3-4. Oocytes of a pre-ovulatory female taken from the batch of lampreys used for field experiments testing putative migratory and mating pheromones. Oocytes displayed were immature, but were in later stages of development than oocytes in Figure 3-3 as they had a nucleus. No columnar follicular cells were present. About 20% of oocytes examined were in this stage of maturity. N = nucleus, Y = yolk, F = follicular cells. Figure presented in color. 160 respectively). During the second trial per night, the proportion of females moving upstream into the bifurcated stream segment did not differ among treatments (Table 2-4). A higher pr0portion of females entered the baited channel during larval extract trials than the proportion entering the baited channel during PADS, PSDS, PZS trials and control trials (Table 2-4 and herein). A higher proportion of females entered within 0.5 m of the larval extract source than those that entered within 0.5 m of sources of PADS, PSDS, PZS and control vehicle. Upstream movement data were analyzed with a mixed effect model (p < 0.001, X2 = 51.51, df = 1). Channel preference and entries within 0.5 m of the odorant source were evaluated with fixed effect models (p > 0.50, df = 1, X2 = -0.01; p > 0.50, df = l, X2 = -0.01; respectfully). During the third trial per night, the proportion of females moving upstream into the bifurcated channels was greater during larval extract trials and 3kPZS trials than during control trials (Table 2-4). The proportion of females moving upstream during larval extract trials and 3kPZS trials did not differ significantly (p = 0.312, df = 1, z-value = 1.01). Female entry into the channel treated with larval extract, 3kPZS, and control vehicle did not differ significantly (Table 3-4 and herein). A higher proportion of females entered within 0.5 m of a source of larval extract than a source of control odorant. Female entry within 0.5 m of a source of 3kPZS did not differ significantly from larval extract and control methanol treatments (3kPZS vs. Extract: p = 0.070, df = 1, z-value = -1.81). Upstream movement, channel preference, and entries within 0.5 m of the odorant source were evaluated with fixed effect models (p = 0.403, df = 1, X2 = 0.70; p > 0.50, df= l, X2 = 0.38; p > 0.50, df= l, X2 = 0.31; respectively). 161 Table 2-4. The number of pre-ovulatory female sea lampreys that were released (11) and the proportion that moved upstream into the bifurcated stream segment when an odorant was applied to one channel and control vehicle was applied to the other, or when control vehicle was applied to both channels (Control Vehicle). Statistical results presented are separated into data collected during the first, second, and third trials per night. A p-value less that 0.05 indicates a significant difference when compared to control vehicle trials. Treatments with the same letter within each group did not differ significantly. Treatment- lst Trial Trials n % Upstream p—value (df, z-stat) Control Vehicle 3 60 67% NA, A Larval Extract 4 80 85% 0.098 (258, l.66)A PADS, PSDS, PZS, 3kPZS 6 120 75% 0.442 (258, 0.77) A Treatment - 2nd Trial Control Vehicle 2 40 38% NA, A Larval Extract 2 40 65% 0.424 (238, 0.80) A PADS, PSDS, PZS 8 160 42% 0.897 (238, 0.13) A Treatment - 3rd Trial Control Vehicle 2 40 25% NA, A Larval Extract 2 40 50% 0.023 (199, 2.27) B 3kPZS 6 120 59% < 0.001 (199, 3.58) B 162 3.1.3.. .3: w: .3 3e 33 w .30.. .3: 9:... 3mm 38 E 3%... m .33 .3: 33: can 33 w .3. .3: 8.... 38 38 2 can: .33 .\ . .2250 E; E». - 2.658; .x .33. .3: 33... ea. 3o. 1:. .3: SN... 3..“ 33 S 3.. .35.. .32.. m .33 .3: 2: 3.. 3% m .33 .3: was 3. m 3% 3 ease. .33.. w ..2 3: 38 < .<2 33 33 m. o.o._.o> .288 3:. E." t 23:53:. 3. .23 .3: 32$ 33 33 m :33 .3: so? 3: 3% 3 3%... .mma .83 .32.. o ..ee... .3: .830. 3. 33 m .3 .m .3: 89$ 33 33 we sex: .23.. w . .eeeeo Outta c3 035?.“ .2250 9:05:52..— Oaumrn ...3 2:57: .530 .9350 .520 ace—=32..— : 3:. .m- ruse—39:. .b.:eocm:w_m 5&6 8: E: 95% some 55.3 5:2 08% on: :33 3:08:35. 23: 23:2, 35:8 88: oofizombv 58$:me a 8:865 36 BE 32 0287: < .EwE 8: 23: BE: :5 6:88 .35 65 wfihfi “088:8 8m: 3:. 388:8 8: 68:82: 3:58 Eoumufim .2838 6:88.053 8:82 ammo—8 288:0 on: :o E m: ESE wEBEo 33:0: E85: :5 .2229? 35:8 38 3330:0882: on: 9.18:0 3.9:“: E35: A5 6089: 885 38833 05 SE 8853.: w:_>oE $0382 mom 2:80: b82398: :0 89:52 dim 035—. 163 Responses to larval extract throughout the night. The proportion of females swimming upstream into the bifurcated stream segment was significantly greater during the first trial per night than the third trial per night (Table 4-4). The proportion of upstream migrantes entering the channel baited with larval extract did not vary significantly depending on whether extract was applied during the first, second, or third trial per night (Table 5-4 and herein). The proportion of females entering within 0.5 m of the larval extract source did not differ significantly among trials conducted during the three time periods. Upstream movement data were analyzed with a mixed effect model (p <0.001, df = 1, X2 = 13.24). Channel preference and entries within 0.5 m of the odorant source were analyzed with fixed effect models (p = 0.113, df = l, X2 = 2.51; p > 0.50, df= 1, X2 < 0.01; respectively). Combined data analysis from all trials. When data from all trials were combined, the proportion of females moving upstream into the bifurcated stream segment during larval extract and 3kPZS trials was significantly greater than the proportion of females moving upstream during control vehicle or PADS, PSDS, PZS trials (Table 4-4; larval extract vs. PADS, PSDS, PZS: p = 0.003, df = 697, z-stat = -2.93; 3kPZS vs. PADS, PSDS, PZS = p = 0.001, df = 697, z-stat = -3.20). The proportion of females moving upstream did not differ between larval extract trials and PADS, PSDS, PZS, 3kPZS trials (p = 0.194, df = 697, z-stat = -l .30), and did not differ between larval extract trials and 3kPZS trials (p = 0.100, df = 697, z-stat = 1.645). Combined upstream movement data were evaluated with a mixed effect generalized linear model, where treatment was a fixed effect and trial date and time were random effects. Trial date was included in the model because likelihood ratio tests showed that trial date was an important random effect in 164 models describing upstream movement within an individual time period. Treatment time was included in the model because it was an important fixed effect influencing upstream movement when all larval extract trials were compared. Larval extract, a mixture of synthesized PADS, PSDS, PZS, 3kPZS, and 3kPZS alone elicited significant preference responses for the baited channel (Table 5-4 and herein). A mixture of PADS, PSDS, PZS did not elicit a significant preference for the baited channel. The proportion of females entering the channel baited with larval extract, PADS, PSDS, PZS, 3kPZS, and 3kPZS alone did not differ significantly. A higher proportion of females entered within 0.5 m of the larval extract source, the mixture of PADS, PSDS, PZS, 3kPZS, and the 3kPZS odorant source than sources of control odorant or a mixture of PADS, PSDS, PZS (PADS, PSDS, PZS vs. Extract: p < 0.001, df = 406, z-stat = 5.39; PADS, PSDS, PZS vs. PADS, PSDS, PZS, 3kPZS: p = 0.10, df= 406, z-stat = 1.66; PADS, PSDS, PZS vs. 3kPZS: p = 0.0321, df = 406, z-stat = 2.14). Larval extract lured significantly more females to within 0.5 m of the pheromone release location than did a mixture of PADS, PSDS, PZS, 3kPZS or 3kPZS alone (p < 0.001, df = 406, z-stat = -4.56; p < 0.001, df = 406, z-stat = -3.66; respectively). Data were analyzed with a fixed effect generalized linear model where variability in channel preference and entry within 0.5 m of the odorant source was explained by treatment. Trial date was not included in the model because likelihood ratio tests within individual time periods showed that trial date was an unimportant random effect describing channel preference and movement within 0.5 m of an odorant source. Treatment time was not included in the model because it did not influence channel preference or movement within 0.5 m of the odorant source when all larval extract trials were compared. 165 Table 4-4. The number of pre-ovulatory female sea lampreys that were released (n) and the proportion that moved upstream into the bifurcated stream segment when an odorant was applied to one channel and control vehicle was applied to the other, or when control vehicle was applied to both channels. Statistical results presented are separated into the data collected during larval extract trials at different times of night and combined data from all trials. A p-value less that 0.05 indicates a significant difference when compared to the first larval extract trial per night (Positive Controls) or control trials (All Trials). Treatments with the same letter within each group did not differ significantly. Positive Controls Trials n °/o Upstream p-value (df, z-stat) Larval Extract lst 4 80 85% NA, A Larval Extract 2nd 2 40 65% 0.155 (158, -1.42) AB Larval Extract 3rd 2 40 50% <0.001 (158, -3.87) B Treatment - All Trials Control Vehicle 8 160 41% NA, A Larval Extract 8 160 71% 0.009 (697, 2.61) BC PADS, PSDS, PZS, 3kPZS 6 120 75% 0.481 (697, 0.705) AC PADS, PSDS, PZS 8 160 42% 0.365 (697, -O.906) A 3kPZS 6 120 59% <0.001 (697, 3.84) B 166 m <2 .m New» Need 30 $3. m .«mefi New» «86 o\omm $3 2. wNn—xm < ANN; .Novv mmmd .ch— $o_ < .8~.o .8”; «.36 $3 $3 no mNm .mem .mgm m .«NAN .33 Sad .Vom .Vowm m .«nmfi .33 Sad Vim $60 ea mNn—xm .mNm .mamm .mgm D .85m New» 396v .x.~ .Vooo m .93. New...» Sedv $2 $2. 3. 823m. 323 V .<2 .3: 30 V . .2280 with =< . 2.25.3.5. < 5.3- ._ _ 3 $3 {an :3. V .32. ._ _ c 9.3. $8 $8 3. E 685 333 V .Amod- ._ 2v 33o .Voo .xcmw V find. ._ _ 5 ~36 .x; m .che 3 EN “owbxm :33.- V .oE 9895: wow ofiEfl bows—«$0-2m go 538: Z 41m 93.; 167 DISCUSSION Upstream movement of pre-ovulatory females into the bifurcated stream segment was influenced by the time of night females were released. During larval extract trials females were more likely to move upstream during the first trial per night. This result is consistent with a study that showed that migratory lampreys are most active during the early portion of the night (Binder & McDonald, 2008). The proportion of females moving upstream during atrial also varied from night to night given the same odorant treatment. Variation in the proportion of females moving upstream on different trial dates and times is likely related to temperature or other stream conditions (Binder & McDonald, 2008; Applegate, 1950). In the analysis of all upstream movement data, a mixed effect model with random effects of trial date and time allowed for the simultaneous evaluation of all odorant treatments by accounting for variation in the data attributed to trial time and date. Pre-ovulatory females displayed similar preference responses to migratory pheromones regardless of the trial time. Females that moved upstream into the bifurcated stream segment showed similar preference responses to larval extract throughout the night. Date of the trial did not influence female entry into the baited channel or entry within 0.5 m of any odorant treatment because the random effect of trial date in mixed effect models did not significantly improve model fit. Given that preference responses of females did not change throughout the night or on different trial dates, all experimental data describing entry into the baited channel and entry within 0.5 m of an odorant source were combined and analyzed with a fixed effect model. 168 A benefit of conducting three trials per night was that changes in female maturity and responsiveness to pheromone treatments over the experimental period were reduced because only 16 days were required to conduct the study rather than 35 days needed if only one trial per night were conducted. Histological analyses of gonads showed that females used in the study were several days from ovulation and the developmental state of the gonads did not change substantially over the duration of the study. Conducting three trials per night did generate the potential confounding effect that behavioral responses of females to odorants may vary through the night. Statistical analyses showed that female preference responses to larval extract did not vary through the night and that behavioral responses to all odorant treatments did not vary among trials. Therefore, the discussion below will focus on analyses of response variables of all combined data. Synthesized 3kPZS influenced the large-scale movement patterns of pre-ovulatory females in a natural stream. 3kPZS alone at 5 x 10'13 M lured more females into the baited channel than the control vehicle. The ability of 3kPZS to lure females into the baited channel of the river did not differ from that of extracted migratory pheromone. 3kPZS also influenced the fine-scale movement patterns of pre-ovulatory females in streams. 3kPZS lured more females within 0.5 m of the odorant release point than the control vehicle. The ability of 3kPZS to lure females to the exact point of release was less than that of larval extract. Larval extract lured about 60% of females that moved upstream within 0.5 m of the odorant application, while 3kPZS only lured about 30% of females to within 0.5 m. Surprisingly, the mixture of putative migratory pheromones, PADS, PSDS, PZS (Sorensen et al., 2005), did not lure females into the baited channel or to within 0.5 m of the odorant release point, unless the mixture was spiked with 3kPZS. 169 Data that demonstrate attraction of pre-ovulatory females to 3kPZS are highly unexpected given the current understanding of sea lamprey chemical communication. In field conditions, pre-ovulatory females have not shown a preference response to 3kPZS (Siefl(es et al., 2005). In a two-choice maze, pre-ovulatory females have not shown a preference response to spermiated male washings which contain 3kPZS (Siefltes et al., 2005). The discrepancies in results are likely attributed to the fact that the current study evaluated behavioral responses at night when pre-ovulatory females migrate, whereas previous studies were conducted during the day. Whether pre-spermiated males show preference responses to 3kPZS remains unknown. In a two-choice maze, pre-spermiated males were not attracted to washings from spermiated males (Sieflo 0:5 :08: 05:: 58553 0:: :o 858% .r L: :52 .m> 35:00 3:883: 5:25:85 5:: 5:: 58:58:: 65:8 8038: 585:8 05... 25:: 55:05.8: 05 :0 85.58: mo5m53m dommmfiwo: 055?: ::3 83396 803 Sun .888 0:5 8 8:39.“. 83 mmem 8:3 85 858980883: 0:: :: 855:8 803 moo—:50: =< .058 mo 2 : m 5: .q 303 m Aoomv < Amx ..«3 cagd A5 m A5 < = with 528950 825 33m nocnouey— 38m .832:me 28:53:. .3552; :8 .1. =3. 28 .mwcfimmz, two: u 982 .mwcfimmB 22: @828..on mo Hombxo D0 203 Sn: 22255me .25me “mo: Em 05 .«o E md :22? €on 08: owmbzw 05 mm: coccouom ABS—co 282.8928 88% 05 E Bum: Efiovo 988m 2: 95858 ..m: was 5:28 acmtamfioa 62% of E Ba: 8825 “mum 2.: 358232 ..<: 2.22.“ m .5 < 23: @225 35 mpg—«Eon no 838:: 05 93 :95: EB :93? .3328 3:25: .«0 58:5: .88 2.: mm 2:: .couombxo 30 was Q 38338 883 Sum .8828 888888 886 85 8 :22— 8828 88% 85 $88.88 am: :5 8828 802-2888 886 85 8 :22— 8808 8.5 86 88888 ..<.. 828888 m _ U 88 goon no $2808 888%: 53, @883 88: oz: 8 @8538 8888.: B888 88 :8 88 888888 88 .8 A5 89828 88:- .m-w via-n. 214 DISCUSSION 3kACA alone, when applied over a thousand-fold range of concentrations did not influence the behavior of ovulated females in streams during the day. 3kACA did not induce females to move upstream over 70 m or 650 m distances. When 3kACA was baited into a trap, the capture rate of ovulated females did not increase. When 3kACA was applied to a nest it did not attract, retain, or induce ovulated female mating behaviors. Behavioral responses of ovulated females to mixtures of 3kPZS and 3kACA did not differ significantly from that of 3kPZS alone. Over a 70 m distance, traps baited with a mixture of 3kPZS and 3kACA captured 64% of females, which was higher than the 46% female capture rate in traps baited with 3kPZS alone observed in a previous study (Chapter 3). However, during 650 m trapping experiments,‘the proportion of females swimming upstream during 3kPZS and 3kPZS + 3kACA treatments did not differ, and the number captured in traps baited with the two test articles did not differ. Additionally, in nesting experiments, a nest baited with 3kPZS alone lured and retained females as well as mixtures of 3kPZS and 3kACA at 1:1 ratios and 1:0.] ratios. Also, a mixture of 3kACA and 3kPZS did not induce more rock movements or tail fans than nests baited with 3kPZS. All field results support the conclusion that 3kACA does not induce behavioral responses in ovulated females. Field results also do not support the hypothesis that minor components of the sea lamprey pheromone may resemble the major pheromone component, as is common in insects (Howse et al., 1998). However, 3kACA may function as a priming pheromone. Recently 3kACA has been shown to slow the 215 maturation of pre-sperrniated males by offsetting the priming response elicited by 3kPZS (Chung-Davison et al., in prep). Spawning channel experiments demonstrated that SMW contains pheromone components in addition to 3kPZS and 3kACA that induce reproductive behaviors in nests. To date, six putative mating pheromone components have been identified using two-choice mazes and only 3kPZS has elicited behavioral responses in natural streams. The lack of success in identifying biologically relevant pheromones highlights the need to reevaluate pheromone extraction techniques and shift bioassays from the lab to the field. The field bioassay developed in this study functioned well to distinguish differences in the behavioral responses of females to two test articles. The evaluation of four response variables at once in the bioassay improved experimental efficiency. The simultaneous evaluation of two test articles can yield clear conclusions on whether female responses to test articles differed. Comparing two odorants at the same time is statistically more powerful than comparing two groups of females that were exposed to different odorants at different times because variability is reduced in the behavioral responses of females and in stream conditions. In most comparisons the bioassay was unbiased; females showed equal preference for the two nests used in the bioassay and were equally likely to be retained and display mating behaviors in each nest. Occasionally, females would prefer the right nest in each channel regardless of the pheromone treatment applied. Right nest biases were accounted for by including the nest effect in the statistical model; however, this reduced experimental power due to a loss of a degree of freedom. XAD7HP resin effectively captured and eluted male mating pheromone components that induced rock movements and tail fans. XAD extract can be confidently 216 probed for unidentified pheromone components because this study confirmed they were present in the extract. Although XAD resin extracted all pheromone components, it did so with low efficiency. In Fine et al. 2006, 3kPZS was extracted with XAD7HP with 81% efficiency. The XAD7HP extraction conducted in this study only yielded 25% of the 3kPZS present in the SMW, although the same equipment was used. Analysis of water that passed through the XAD resin showed that 25% of 3kPZS in the SMW was present in the waste water. Bioassay comparisons determined that unidentified pheromone components were also in the waste water at high enough quantities to elicit behavioral responses. Pheromone components may have passed into the waste water due to poor packing of XAD resin in the glass column. Poor packing would result in channelization of water through the column reducing resin surface area and the time pheromones were exposed to the resin. Using more XAD resin and slower flow rates would likely improve extraction efficiency. 50% of 3kPZS was likely retained on the resin and not eluted when washed with 100% methanol because it was not present in the extract or waste water. Rinsing the resin with more than 3 L of methanol may improve elution efficiency. Alternatively, stronger solvents could be used to increase XAD elution efficiency. C18 sep-paks extracted and eluted 3kPZS from XAD extract with high efficiency, but did not contain behaviorally active quantities of pheromone components that induce retention and mating behaviors in a nest. Surprisingly, the field bioassay showed that C18 resin likely captured unidentified pheromone components that induced mating behaviors, but did not release them when the resin was rinsed with 100% methanol. Li et a1. 2002 likely had excellent success isolating and purifying 3kPZS from SMW because 217 they employed C18 sep-paks. Ironically, attempts to identify additional pheromone components were hampered by employing C18 sep-paks because they were not present in the extract. A main conclusion of this study is that C18 resin should not be used to concentrate XAD extract of spermiated male washings when searching for unidentified pheromone components. Bioassay comparisons of head and tail washing demonstrated that all behaviorally active pheromone components were excreted through the head region. These results confirmed the findings of SieflPetromyzon marinus Type Of Use: Book / Textbook Total: 0.00 USD To access your account, please visit https://myaccount.copyright.com. Please take a moment to complete our customer satisfaction survey. http://www.surveymonkcy.com/s.asp?u=500021004336 If you have any comments or questions, please contact Rightslink: Copyright Clearance Center Rightslink Tel (toll free): 877/622-5543 Tel: 978/777-9929 E-mail: mailto:customercare@copvright.com Web: MJ/wwwcopyrightcom B.1:v4.1 ELSEVIER LIMITED LICENSE TERMS AND CONDITIONS 227 Apr 18,2008 __._— --__.___—_-.. ‘ I-~“—— - ._--L _h _: ,_ A ,_ 7 _. .—._.._.___.-~..--__——_—-- _. ‘—- .._.__.‘. _____ ..- - “H... .. .. ~ ..-- ._.—-_.’.-.._ ,-_.-...._.. This is a License Agreement between Nicholas S Johnson ("You") and Elsevier Limited ("Elsevier Limited"). 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