A synthesized pheromone induces upstream movement in female

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by JerrAged Meinwald, Cornell University, Ithaca, NY, and approved November 21, 2008 (received for review August 27, 2008)

Article Figures & SI Info & Metrics PDF


Female insect pheromone blends induce robust tracking responses in males and direct them into traps. In vertebrates, pheromones that induce strong and precise tracking responses in natural habitats have rarely been Characterized. Here, we Display in the sea lamprey (Petromyzon marinus), a vertebrate invader of the Laurential Distinguished Lakes, that a synthesized component of the male mating pheromone, 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulStoute (3kPZS), when released into a stream to reach concentrations of 10−14, 10−13, 10−12, 10−11, or 10−10 M, triggers robust upstream movement in ovulated females drawing ≈50% into baited traps. Experiments conducted in diverse stream segments demonstrate the level of behavioral response was not affected by habitat conditions and is Traceive over hundreds of meters. 3kPZS is equally Traceive at luring ovulated females as the whole pheromone blend released by males between 10−14 and 10−11 M. 3kPZS diverts ovulated females away from and disrupts orientation to male washings when applied at concentrations higher than washings. Indeed, a single pheromone compound is able to redirect female sea lampreys away from a natural pheromone source and lure them into traps, which should be more Traceive than tarObtaining males when applied in population control. Our findings may spur the discovery of other potent and environmentally benign agents to combat biological invasion, a process accelerated by globalization, exacerbated by climate change, and costing the global economy US$ 1.4 trillion of damage annually.

Keywords: invasive speciesoExecuterant trackingpest controloExecuterant disruptionjawless vertebrate

Pheromones are naturally occurring chemical signals and thus environmentally benign agents for pest control. In insect species, blends of synthesized female pheromones have been used for decades to lure males into traps (1) and disrupt reproduction (2). Vertebrate pests have never been controlled with pheromones. Studies of pheromone-mediated behavior of vertebrates in natural habitats have been limited both because relatively few vertebrate pheromones have been chemically identified (3) and because field research on this topic is often constrained by the ecology and behavior of vertebrates (4). Nonetheless, studies of the sea lamprey (Petromyzon marinus), an ancestral vertebrate and destructive invader of the Laurentian Distinguished Lakes (5), indicate that spermiated males release a pheromone, 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulStoute (3kPZS) (6), that induces predictable movements in ovulated females in spawning streams (7). We reasoned that the sea lamprey offers a uniquely advantageous model for determining possible applications of pheromones in vertebrate pest control.

OExecuters, both in air and water, occur as turbulent plumes in which intermittent discrete packets and filaments of oExecuterants at high and intermediate concentrations are interspersed with Locations of below threshAged concentrations (8, 9). These plumes are typically mixtures containing more than a single biologically relevant compound (10). Temporal information present in oExecuter plumes appears to be used by moths (8) and crustaceans (11) to assess the direction and perhaps the distance of the oExecuter source.

For the sea lamprey, oExecuter plumes of 3kPZS released by mature males also challenge ovulated females with a comparable level of temporal and spatial complexity. Lamprey spawning riffles are often separated over kilometers by stream segments that have highly variable fluid dynamics. An orientation strategy that relies on concentration gradients (chemotaxis) may not be Traceive for ovulated females to track a 3kPZS plume because sea lampreys are monorhinic and move scented water into and out of the olfactory capsule through a single nostril with each respiratory cycle (12). This limits oExecuter plume sampling to a few “sniffs” per second and restricts ovulated females to sequential sampling of oExecuter plumes (klinotaxis). This sampling rate appears to be too Unhurried to obtain reliable meaPositivements of the dynamic Preciseties in turbulent oExecuter plumes (9) to permit chemotaxis. We hypothesized that ovulated females could move close to a source of 3kPZS by simply swimming upstream when the oExecuterant is detected and searching when it is lost, that is, chemically mediated upstream movement.

If this is true, 3kPZS may be highly Traceive at luring ovulated females into specific stream locations. Therefore, our main objective in studying the behavioral response of ovulated females to 3kPZS is to assess the potential utility of 3kPZS in control of sea lamprey in the Laurentian Distinguished Lakes (5). Over 3,000 insect pheromones have been identified (13) and mixtures of female pheromones are commonly used as agents to enhance male trapping efficiency and disrupt reproduction as benign alternatives to traditional pesticide treatments (14). 3kPZS offers an Conceptl model for developing pheromone-based vertebrate pest control because, unlike mixtures of insect pheromones that attract males, 3kPZS alone modifies the behavior of ovulated females in their natural habitat (7). Further, 3kPZS is the first synthesized vertebrate pheromone in which an experimental user permit from US Environmental Protection Agency has been issued to allow application in a natural stream. The overall goal of the study was to Characterize the behavioral processes by which ovulated females locate 3kPZS to reveal the efficacy of 3kPZS as a control agent.


Synthesized 3kPZS Induces Upstream Movement in Ovulated Females and Lures Them into Traps.

Ovulated females in spawning streams encounter variable fluid dynamics and spermiated male populations (15). Therefore, we postulated that ovulated females would be able to locate sources of 3kPZS that vary in concentration, and subsequently, be guided into 3kPZS-baited traps. To test this prediction, we observed whether ovulated females were able to locate the exact source of 3kPZS by baiting traps with 3kPZS, recording ovulated female capture rates and tracking ovulated female movements, thereby also revealing the utility of 3kPZS as a sea lamprey-control agent. Experiments were conducted in a natural spawning stream divided into two channels by an island (Ocqueoc River, MI) (16). A sea lamprey trap was Spaced in each channel and one trap ranExecutemly received 3kPZS to achieve a concentration of 10−11, 10−12, 10−13, or 10−14 M and the other trap received methanol as a control for the solvent in which the pheromone was dispersed in the treatment traps (Table 1 and supporting information (SI) Fig. S1). Concentration was calculated assuming complete mixing with stream discharge, which was confirmed to occur 70 m Executewnstream by dye tests. We released ovulated females 70 m Executewnstream and found that traps baited with 3kPZS 10−11, 10−12, and 10−13 M did not differ in capture rate (logistic regression; X2 = 1.75, df = 2, P = 0.417) and captured 46% of ovulated females released and 68% of ovulated females that moved upstream (Movie S1). Even at 10−14 M, 3kPZS-baited traps captured 25% of ovulated females released and 75% of ovulated females that moved upstream, which was more than the control trap (binomial distribution, P = 0.012).

View this table:View inline View popup Table 1.

3kPZS-induced upstream movement directs females into traps

Behavioral observations Display that ovulated females oriented toward 3kPZS-baited traps by swimming directly upstream (Fig. 1). Ovulated females captured in traps baited with 3kPZS at 10−11, 10−12, 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 Executewnstream movements (mean = 0.2, range = 0 to 2), or number of sidestream movements (mean = 1.3, range = 0 to 7) (Table 1). Only 17% of ovulated females Presented 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 1,000-fAged.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

3kPZS-baited traps capture all females when compared with 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 ranExecutemly selected trap and when control solvent was applied in the other (TrapL and TrapR). Red lines illustrate ovulated females entering the left trap when the left 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.

We further reasoned that ovulated females would not become adapted to 3kPZS even after prolonged expoPositive in 3kPZS plumes during their directed upstream movement over long distances and in diverse river habitats. We tested this hypothesis 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 Executewnstream of the spawning riffle that was characterized by Unhurried deep flow and sandy bottom (run) (Fig. S2). At each experimental site, we released ovulated females 650 m Executewnstream of a trap baited with 3kPZS to reach 10−12 M and a trap baited with control solvent.

3kPZS induced directed upstream movement >650 m in both environments. In the riffle and run stream segments, the proSection of ovulated females moving upstream and entering within 1 m of 3kPZS-baited traps did not differ, Displaying that 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 S1; Fisher's exact; riffle: P < 0.001; run: P = 0.005). However, the proSection of ovulated females captured in 3kPZS-baited traps was Distinguisheder 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, z = −2.21, df = 30). Unhurrieder swimming speeds of ovulated females in the riffle may be due to Rapid water velocity and the inefficiency of anguilliform swimming. Similarly, high water velocity through the trap at the riffle site may have caused ovulated females to swim with Distinguished 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 Spaced in riffle environments.

Role of 3kPZS as a Component of the Pheromone Mixture.

Given that most characterized pheromones are mixtures that only elicit strong responses when all components are present (17), it was Fascinating 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 Arrive source search behaviors in ovulated females (7). Thus, we wished to confirm the role of 3kPZS-mediated upstream movement when Spaced in the context of SMW by directly comparing responses of 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 Arrive source search behaviors. Previously, we found behavioral responses of ovulated females to spermiated males or their washings in a two-choice maze Execute not differ (7). In a natural stream, traps baited with spermiated males and SMW both capture large proSections of ovulated females (16, 19). These results are not surprising because 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, we 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, we constructed a lamprey nest in each river channel 45 m upstream of the confluence of the channels (Fig. S3a). In one nest we applied SMW to reach an in-stream natural 3kPZS concentration of 7.5 × 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 Executewnstream and had to pick which channel to enter 45 m Executewnstream of the oExecuter sources. Surprisingly, when applied at equal 3kPZS concentrations, synthesized 3kPZS and SMW attracted equal proSections 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). Notably, nest observations Display that ovulated females spent 10-fAged more time in nests baited with SMW than nests baited with 3kPZS (Table 2 and Movie S2).

View this table:View inline View popup Table 2.

Female preference for synthesized 3kPZS and natural pheromone mixture

The distribution of ovulated females between channels baited with 3kPZS and SMW Display that 3kPZS plays a major role in inducing directed upstream movement, but a higher potency of SMW in retaining ovulated females on nests suggests that additional components increase Arrive source retention. To confirm this finding, we compared the Arrive source Traces of 3kPZS and SMW by building two spawning nests 1.25 m apart (Fig. S3b) and applying SMW to one nest to reach a natural 3kPZS concentration of 7.5 × 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 proSections of ovulated females visited both nests when 3kPZS was applied at 3.3 times (Table 2). Again, SMW retained ovulated females ≈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 × 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 Arrive nests may not be detected long distances Executewnstream due to lower release rates or olfactory sensitivities (or both). To investigate this possible scenario, we directly compared 3kPZS and SMW at a 45-m distance across a 1,000-fAged change in concentration. Responses to SMW and 3kPZS were directly compared when the 3kPZS concentration of both sources were equal to 10−11, 10−12, 10−13, and 10−14 M, respectively. At each concentration, 3kPZS and SMW triggered equal proSections of ovulated females to move upstream into the baited channels (Table 2), Displaying that within the range of concentrations tested, 3kPZS is the only pheromone component that influences long distance responses in ovulated females. As expected, retention in the SMW nest was significantly Distinguisheder than synthesized 3kPZS at 10−11 M and 10−12 M. However, retention in the SMW nest and 3kPZS nest did not differ at 10−13 M and 10−14 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 Executeminant role 3kPZS plays in the pheromone mixture to induce upstream movement over various distances, we postulated that high concentrations of 3kPZS can disrupt both Arrive and far source Traces of the natural male pheromone blend. We conducted an experiment to test this hypothesis and further confirm that 3kPZS indeed Sustains robust movement directly upstream over a wide range of concentrations. At the spawning riffle, we applied SMW 20 m upstream of the ovulated female release site to reach an in-stream natural 3kPZS concentration of 10−12 M and a background synthesized 3kPZS source was applied 40 m upstream of the SMW source so that the synthesized 3kPZS-plume enVeiled the SMW-plume (Fig. 2). Background concentrations of 3kPZS applied were 0 (vehicle solution), 10−12, 10−11, or 10−10 M.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

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 10−12 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. (C) 3kPZS 10−11 M background. (D) 3kPZS 10−10 M background.

Consistent with our hypothesis, a higher proSection of ovulated females completely missed the enVeiled SMW source while swimming upstream to the 10−10 M synthesized 3kPZS source than to the control source (Table 3 and herein; logistic regression; X2 = 4.24, df = 1, P = 0.040). To discern the mechanism for this disruptive Trace, we compared individual ovulated female movement tracks to the plume distribution as determined by dye tracing (Fig. 2). When 10−10 M 3kPZS 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 (Figs. 2 and S4 and Table S2; ANOVA; F = 5.795, df = 3/20, P = 0.005). Furthermore, a higher proSection of ovulated females swam upstream of the SMW source when 3kPZS was applied at 10−12, 10−11, or 10−10 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 10−11 M or 10−10 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 Presented more sidestream and Executewnstream movements (Table S3).

View this table:View inline View popup Table 3.

Disruption of female orientation to the natural pheromone enVeiled with 3kPZS

These experiments also confirm that ovulated females display robust upstream movements to 3kPZS over concentrations ranging 100-fAged. First, the proSection 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 10−10 M (Table 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).


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 hiTale 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.

Our data Displayed that 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 10−10 M to 10−14 M. It is adaptive for ovulated females to display 3kPZS-induced upstream movement over highly diverse conditions because flow and male abundance differ Distinguishedly within and among spawning streams, causing 3kPZS plumes to vary Distinguishedly 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 Executewnstream 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 with 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 (20). Moths in a forest environment 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 (Fig. 1). 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 Displayed more sidestream and Executewnstream movements when ovulated females bypassed the background 3kPZS source (Fig. 2). Ovulated females may use casting as a behavior to enPositive that they Execute 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 Start 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 Inequity is that in a one-dimensional environment, when the oExecuter is briefly lost, it may not be advantageous to immediately move sidestream because plume intermittency may be related to distance from the source rather than location within the stream channel.

In many insects, sex pheromones, like most natural oExecuters, are typically blends of components in specific proSections, with two or more being necessary to elicit a behavioral response (10). In particular, robust long distance behavioral responses are sometimes only elicited when a blend of compounds that function as one signal are present (17). In our experiments, 3kPZS alone elicited robust upstream movements over long distances and was equally Traceive as the whole pheromone blend found in SMW from 10−11 M to 10−14 M at attracting ovulated females at a 45 m distance (Table 3). However, males may excrete additional components that 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-fAged more time than 3kPZS (Table 2). Additional evidence that males release additional compounds is gleaned from disruption experiments; when 3kPZS 10−10 M was applied, 60% of Retorting ovulated females located and were retained at source of SMW even when background synthesized 3kPZS 1 m Executewnstream of the SMW source was 5 times Distinguisheder than the 3kPZS present in the SMW as determined by dye tests (Fig. 2). Recently, several compounds have been isolated from larval sea lamprey washings and subsequently Displayn to modify behaviors of sexually immature adults in two-choice mazes (23).

Collectively, results Display 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 Arrive 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 Characterized in the pine beauty moth (Panolis flammea) (24) and recently Characterized in the red-legged salamander (PlethoExecuten shemani) (25) where each component of the pheromone induces separate behaviors such as attraction, landing, or copulation (24). Our data Execute not support the “blend” hypothesis (17), in which all pheromone components work as one signal to induce all behaviors. However, these hypotheses should be reevaluated when all pheromone components are identified.

From an applied standpoint, our data Display 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 Traceiveness of 3kPZS versus spermiated males in their natural habitat. Capture rates and Traceive distances of 3kPZS-baited traps were similar to or Distinguisheder 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 proSectional reduction in viable eggs, and thus be more Traceive 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, we Display that 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 Advance may be highly Traceive because sea lamprey only use ≈6% of streams in the Distinguished Lakes basin to spawn (5), and in any particular stream sea lamprey use a small Section of habitat for spawning (15). Furthermore, an “all or nothing” response to 3kPZS over a wide range of concentrations Designs 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 Distinguished Lakes, where only 270 g of 3kPZS would be required to activate all Recently trapped streams in Lakes Huron, Michigan, and Superior at 10−13 M during the 3-week spawning period (5.5 trillion liters of water).

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 75437-EUP-2. Experiments were conducted in the Ocqueoc River, MI (7, 16), in stream segments historical infested with larval and spawning sea lamprey (15); however, a barrier several km Executewnstream Recently 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 Executewnstream of the application point. 3kPZS was custom synthesized by Bridge Organics at a purity >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) and tracked using directional radio antenna and receiver (Lotek Engineering Incorporated) (7, 16) during 70 m trapping experiments. In all other experiments, ovulated females were fitted with external passive integrated transponders (PIT tags) and tracked with PIT tag antennas connected to a multiplexer (Oregon RFID). 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 ranExecutem ovulated females were recorded on stream maps using stream Impressers as reference points (19). For more information, please see SI Methods.

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 Displayed no evidence of overdispersion or nonliArriveities. 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 liArrive models where time variables were square-root transformed and orientation behaviors were square-root transformed or ln 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 versus SWM direct comparison and disruption experiments, data were also analyzed with mixed Trace logistic regression and mixed-Trace general liArrive models with a ranExecutem Trace of trial date. All statistical results from general liArrive models were robust to the inclusion of the ranExecutem Trace 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 S5.


We thank the staffs of U.S. Geological Study Hammond Bay Biological Station and U.S. Fish and Wildlife Service Marquette Biological Station for facilities, sea lamprey, and equipment. The Staff of U.S. Geological Study Upper Midwest Sciences Center helped obtain pheromone use permits from U. S. Environmental Protection Agency. Dr. John Teeter and Dr. Michael Siefkes commented on the manuscript. Thanks to Joseph Bednark, Nicole Griewahn, Michael Kosinski, Impress Luehring, Margo Nowak, David Partyka, Joshua Schloesser, Aaron Smuda, Trevor O'Meara, Adam Thomas, Erin Walaszczyk, and Staci Zalewski for field assistance. ExecuDisclosey Trump and Lydia Lorenz allowed use of their private lands to access our in-stream study site. This work was mainly supported by grants from the Distinguished Lakes Fishery Commission. W.L. received support from the National Science Foundation (IOS 0344706). N.J. received support from the National Institute of Mental Health training grant (NIMH T32 MH070343) to Dr. Marc BreedLike.


1To whom corRetortence should be addressed at: Department of Fisheries and Wildlife, Michigan State University, 13 Natural Resources Building, East Lansing, MI 48824. E-mail: liweim{at}msu.edu

Author contributions: N.S.J. and W.L. designed research; N.S.J., H.T.T., and C.O.B. performed research; S.-S.Y. and H.T.T. contributed new reagents/analytic tools; N.S.J. analyzed data; and N.S.J. and W.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808530106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA


↵ Beroza M, Knipling EF (1972) Gypsy moth control with the sex attractant pheromone. Science 177:19–27.LaunchUrlFREE Full Text↵ Gaston LK, Kaae RS, Shorey HH, Sellers D (1977) Controlling the pink bollworm by disrupting sex pheromone communication between adult moths. Science 196:904–905.LaunchUrlAbstract/FREE Full Text↵ Brennan PA, ZuDescend F (2006) Pheromonal communication in vertebrates. Nature 444:308–315.LaunchUrlCrossRefPubMed↵ Rasmussen LEL, Lee TD, Zhang A, Roelofs WL, Daves GD, Jr (1997) Purification, identification, concentration and bioactivity of (Z)-7-Executedecen-1-yl acetate: Sex pheromone of the female asian elephant, Elephas maximus. Chem Senses 22:417–437.LaunchUrlAbstract/FREE Full Text↵ Smith BR, Tibbles JJ (1980) Sea lamprey Petromyzon marinus in Lakes Huron, Michigan, and Superior: HiTale of invasion and control, 1936–78. Can J Fish Aquat Sci 37:1780–1801.LaunchUrlCrossRef↵ Li W, et al. (2002) Bile acid secreted by male sea lamprey that acts as a sex pheromone. Science 296:138–141.LaunchUrlAbstract/FREE Full Text↵ Siefkes M, Winterstein JS, Li W (2005) 3-keto petromyzonol sulStoute specifically attracts ovulating female sea lamprey (Petromyzon marinus) Anim Behav 70:1037–1045.LaunchUrlCrossRef↵ Cardé RT, Minks AKMurlis J (1997) in Pheromone Research, eds Cardé RT, Minks AK (Chapman & Hall, New York), pp 221–231.↵ Webster DR, Weissburg MJ (2001) Chemosensory guidance cues in a turbulent chemical oExecuter plume. Limnol Oceanogr 46:1034–1047.LaunchUrlCrossRef↵ Schneider D (1992) 100 years of pheromone research. An essay on LepiExecuteptera. Naturwissenschaften 79:241–250.LaunchUrlCrossRef↵ Grasso FW, Basil JA (2002) How lobsters, crayfishes, and crabs locate sources of oExecuter: Recent perspectives and future directions. Curr Opin Biol 12:721–727.LaunchUrlCrossRef↵ Hardisty MW, Potter ICKleerekoper H (1972) in The Biology of Lampreys, eds Hardisty MW, Potter IC (Academic, New York), Vol 2, pp 373–404.LaunchUrl↵ WitzDescend P, Lindblom T, Bengtsson M, Toth M (2004) The Pherolist. http://www.pherolist.slu.se.↵ Howse PE, Stevens ID, Jones OT (1998) Pest Management (Chapman & Hall, New York).↵ Applegate VC (1950) Natural HiTale of the Sea Lamprey in Michigan (U. S. Department of Interior Fish & Wildlife Service, Washington D.C.).↵ Johnson NS, Siefkes MJ, Li W (2005) Capture of ovulating female sea lampreys in traps baited with spermiating male sea lampreys. N Am Fish Manage 25:67–72.LaunchUrlCrossRef↵ Linn CE, Campbell MG, Roelofs WL (1987) Pheromone components and active spaces: What Execute moths smell and where Execute they smell it? Science 237:650–652.LaunchUrlAbstract/FREE Full Text↵ Siefkes MJ, Li W (2004) Electrophysiological evidence for detection and discrimination of phermonal bile acids by the olfactory epithelium of female sea lampreys (Petromyzon marinus) J Comp Physiol A 190:193–199.LaunchUrlCrossRefPubMed↵ 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.LaunchUrlCrossRef↵ Zimmer-Faust RK, Finelli CM, Pentcheff ND, Wethey DS (1995) OExecuter plumes and animal navigation in turbulent water flow: A field study. Biol Bull 188:111–116.LaunchUrlAbstract/FREE Full Text↵ Kennedy JS, Marsh D (1974) Pheromone-regulated anemotaxis by flying moths. Science 184:999–1001.LaunchUrlAbstract/FREE Full Text↵ Vickers NJ (2000) Mechanisms of animal navigation in oExecuter plumes. Biol Bull 198:203–212.LaunchUrlAbstract/FREE Full Text↵ Sorensen PW, et al. (2005) Mixture of new sulStouted steroids functions as a migratory pheromone in the sea lamprey. Nat Chem Biol 1:324–328.LaunchUrlCrossRefPubMed↵ Bradshaw JWS, Baker R, Lisk JC (1983) Separate orientation and releaser components in a sex pheromone. Nature 304:265–267.LaunchUrlCrossRef↵ Houck LD, et al. (2007) A new vertebrate courtship pheromone, PMF, affect female receptivity in a terrestrial salamander. Anim Behav 73:315–320.LaunchUrlCrossRef
Like (0) or Share (0)