Behavioural responses of female lake trout Salvelinus namaycush to male chemical stimuli and

prostaglandin F2α

Tyler J. Buchinger1, Weiming Li1, Nicholas S. Johnson2*

1
Department of Fisheries and Wildlife, Michigan State University, East Lansing MI, USA
2
Hammond Bay Biological Station, Great Lakes Science Center, U.S. Geological Survey,
Millersburg MI, USA

*Author for correspondence. Hammond Bay Biological Station, 11188 Ray Road, Millersburg,
MI 49759 USA. nsjohnson@usgs.gov

Funding information: The Great Lakes Fisheries Commission and the Great Lakes Fishery Trust
provided funding.

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/jfb.14446

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Abstract
Male olfactory cues may guide aggregation on spawning reefs, mate evaluation, and
synchronized gamete release in lake trout Salvelinus namaycush, but a lack of information on the
source and identity of the cues precludes direct tests of their function. Using a two-channel
flume, we found ovulated female lake trout increased time spent in the channel treated with
spermiated male-conditioned water, urine, and bile but not synthesized prostaglandin F2α. We
suggest future efforts to characterize male olfactory cues focus on urine and postulate that bile
acids contribute to its behavioural activity.

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Key words: Bile acids, chemical ecology, communication, olfactory cues, urine
Lake trout Salvelinus namaycush (Walbaum 1792) are an ecologically diverse keystone species

with great economic and cultural importance (Muir et al., 2013). Genetically distinct populations

are adapted to different ecological niches and likely in early stages of speciation. Valuable

fisheries once targeted lake trout populations throughout the Laurentian Great Lakes, but

overfishing, habitat degradation, and sea lamprey (Petromyzon marinus) predation drove most to

near extirpation (Muir et al., 2013). Rehabilitating lake trout in the Great Lakes and, more

broadly, understanding the mechanisms underlying their diversity is hindered, in part, by a lack

of information on the cues that guide reproduction (Muir et al., 2012).

Olfactory cues may guide aggregation on spawning reefs, mate evaluation, and synchronized

gamete release in lake trout (Muir et al., 2012). Most lake trout spawn over offshore reefs during

autumn (Esteve et al., 2008; Muir et al., 2012; Binder et al., 2015). Males are the first to arrive

on reefs and may signal to females on the periphery of spawning grounds before spawning

begins. Later, a female and several males travel together before simultaneously sinking to the

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lake bottom and releasing gametes (Esteve et al., 2008; Muir et al., 2012; Binder et al., 2015).

Previous studies indicate odours from sexually mature males (spermiated) and females

(ovulated) attract mature conspecifics (Buchinger et al., 2015). Male odours appear to be

especially important; females and males are strongly attracted to male odour, but males, and not

females, exhibit only weak attraction to female odour (Buchinger et al., 2015). Defining the

specific functions of male olfactory cues requires information on their source and identity.

We tested behavioural responses of ovulated female lake trout to male chemical stimuli and a

putative pheromone component. Stimuli tested included (1) male-conditioned water as a positive

control, (2) male urine, which is often a major source of olfactory cues (Stacey, 2015), (3) male

bile, because previous studies on lake trout implicate bile acids as potent olfactory cues (Zhang

et al., 2001), and (4) synthesised prostaglandin F2α (PGF 2α), which functions as a male

pheromone in closely related Arctic char (S. alpinus; Sveinsson and Hara, 1995) and is a potent

olfactory stimulant in lake trout (Hara and Zhang, 1998).

Details regarding experimental animals are reported in Buchinger et al. (2015), which described

studies on the same group of fish. Briefly, age 9-10 female Seneca Lake strain lake trout (n = 21;

71.42 ± 0.9 cm, 3.94 ± 0.34 kg; mean ± SE; biological data missing for two fish) were implanted

with passive integrated transponder tags (PIT; Oregon RFID, Portland, Oregon) and studied at

the United States Geological Survey’s Great Lakes Science Center, Hammond Bay Biological

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Station. All experiments were approved by the Michigan State University Animal Use and Care

Committee (AUF No. 08/12-148-00).

Behavioural responses to chemical stimuli were determined using a flow-through flume with two

channels (Figure 1a). The flume was supplied with ambient Lake Huron water originating from

an intake 25 m deep. The channels were separated by an aquarium holding a spermiated male to

provide a visual cue; the effluent of the aquarium was pumped out of the assay unless male-

conditioned water was being tested. A trial began when an ovulated female was placed in an

acclimation area sectioned off the most downstream area by a removable mesh gate. After 30

min, the fish was released to explore the flume without any stimulus being pumped. After a 30-

min control period, the focal stimulus and appropriate control were pumped into the channels.

The stimulus was allowed to disperse through the channels for 10 min, after which females were

allowed to explore the flume for another 30 min. The time a female spent in a channel was

determined using a PIT array that included an upstream and downstream antenna in each

channel. Trials were excluded if fish did not spend at least 5 s in each channel during the pre-

stimulus period. The side that received the stimulus was alternated each trial. Eight fish were

mistakenly tested with the same stimulus twice (male, urine, PGF2α); data for these fish were

averaged across the trials to avoid pseudoreplication. The time spent in the control and

experimental channels before (bc, be) and after (ac, ae) stimulus application were used to

calculate an index of preference for each channel (control = ac/[ac + bc]; experimental = ae/[ae +

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be]). The indices were compared using Wilcoxon signed-rank tests (α = 0.05; Li et al., 2002).

Trials were conducted at night 3 - 24 November 2013, during which temperatures ranged from

9.8 °C down to 3.4 °C.

Stimuli were applied using a peristaltic pump (Masterflex L/S; www.coleparmer.com). The

aquarium holding a male was supplied with 500 mL min-1 ambient Lake Huron water and the

effluent of the tank was applied as the male-conditioned water. Urine was collected according to

Yambe et al. (1999). Spermiated males (n = 2) were anesthetized using 0.08% (by volume) clove

oil and a 2 mm (external diameter) tubing inserted into the urinary bladder and secured to the

anal, pelvic, and dorsal fins. The fish were placed in a 200 L aquarium and urine collected in

beakers held on ice. Urine was frozen each day over 3 days for a total of 210 mL. Each trial in

which responses to urine were tested used 10 ml. Bile was collected from the gall bladder using a

syringe and 100 µL applied per trial. Synthesized PGF2α from Cayman Chemical

(www.caymanchem.com; CAS 551-11-1) was applied to reach a concentration of 10-10 or 10-11

M using the total volume of the flume. The control treatment was the solvent (50 % methanol;

v:v) for PGF2α and Lake Huron water for the other stimuli. Urine, bile, and PGF2α were mixed

into 8 L of Lake Huron water and applied at a rate of 200 mL min-1.

Our results confirmed female lake trout attend to chemical stimuli release by males and indicate

that attractive compounds are present in male bile and urine but do not include PGF2α (Figure

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1b). Across all trials, females spent 294.6 ± 28.2 s in the left channel and 331.3 ± 35.1 s in the

right channel during the control period (mean ± se, n = 49). In male-conditioned water trials,

females spent 569.7 ± 73.7 s in the experimental channel versus 213.0 ± 45.3 s in the control

channel (n = 10). For male urine trials, females spent 511.7 ± 141.8 s in the experimental channel

versus 204.1 ± 58.9 s in the control channel (n = 10). Females spent 655.4 ± 163.9 s in the

channel treated with male bile versus 190.6 ± 54.9 s in the control channel (n = 9). Females spent

371.0 ± 78.8 s in the channel activated with 10-11 M PGF2α versus 433.2 ± 119.8 s in the control

channel (n = 10) and 271.0 ± 45.2 s in the channel activated with 10-10 M PGF2α versus 370.1 ±

89.4 s in the control channel (n = 10). Based upon these results, we suggest future efforts to

characterize male odour focus on urine and postulate the odour consists, in part, of bile acids.

How bile acid mating pheromones evolve is puzzling given they lack an intuitive link to

reproduction (Buchinger et al., 2014). The release of hormonal pheromones, for example,

coincides with specific physiological events within an animals reproductive system (Stacey,

2015). In contrast, fish primarily excrete bile acids via faeces and as a result of digestion.

Nevertheless, some fish respond to bile and bile acids as mating pheromones (Buchinger et al.,

2014). One explanation of why a male pheromone might consist of bile acids in lake trout is it

matches a juvenile cue that indicates habitat fit for spawning and offspring survival (Buchinger

et al., 2015). Spawning lake trout orient towards juvenile odour (Buchinger et al., 2015;

Buchinger et al., 2017) and spawn over reefs treated with fry faeces (Foster, 1985), which

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contain bile acids that are potent olfactory stimuli (Zhang et al., 2001). However, the juvenile

cue is unlikely useful for spawning site selection for most populations (Buchinger et al., 2017),

in which juveniles leave spawning reefs in early summer (Martin, 1957; Deroche, 1969) and

adults spawn in autumn (Esteve et al., 2008; Muir et al., 2012; Binder et al., 2015). Males may

assume this role of juveniles by releasing some of the same compounds in juvenile odour;

indeed, male lake trout orient towards male and juvenile odour but do not discriminate between

the two (Buchinger et al., 2015). Our observation that females orient towards male bile lends

further support to this hypothesis as bile acids are likely components of juvenile odour (Zhang et

al., 2001), though bile may contain odourants other than bile acids. Interestingly, Zhang (1996)

reported higher concentrations (approximately 100 x) of bile acids in urine from a pre-spawning

male (1-2 week pre-spawn) than juveniles or a female. Additional studies are needed to

determine the function and evolution of female preference for bile.

The indifference of female lake trout to PGF2α suggests a notable divergence from closely related

Arctic char, which use PGF2α as a male sex pheromone that evokes spawning behaviours in

females (Sveinsson and Hara, 1995). We used an assay similar to previous experiments on Arctic

char and tested the same concentration of PGF2α (Sveinsson and Hara, 1995). Why lake trout

might respond to PGF2α differently from Arctic char remains unclear, though the two species

exhibit several differences in their spawning behaviour; for example, female lake trout do not

construct nests (Esteve et al., 2008) and males do not aggressively guard females or territories

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from other males (Muir et al., 2012; see Brattli et al., 2018 for description of Arctic char

spawning behaviour). Notably, female lake trout may follow males to spawning reefs but males

follow females immediately prior to the act of spawning (Muir et al., 2012). Therefore, females

rather than males may be the source of hormonal pheromones, such as PGF2α, that guide

synchronized gamete release. Indeed, PGF2α functions as a female sex pheromone that stimulates

male spawning behaviour in goldfish and likely many other species (Stacey, 2015). Future

research should investigate the potential role of PGF2α as a female sex pheromone.

Acknowledgements

The US Fish and Wildlife Service Sullivan Creek National Fish Hatchery provided fish. Skye

Fissette, Carrie Kozel, and Melissa Pomranke assisted with experiments. Jacob Kimmel, Peter

Hubbard, and three anonymous reviewers provided useful feedback. Use of trade, firm, or

product names is for descriptive purposes only and does not imply endorsement by the US

Government. The Great Lakes Fisheries Commission (2011_JOH_4415) and the Great Lakes

Fishery Trust (#2016.1631) provided funding.

Contributions

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T.J.B. conducted experiments, analyzed data, and drafted the paper. All authors contributed to

developing the ideas, securing funding, and editing the paper.

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 Figure 1. a) Schematic of apparatus used to test preferences for natural and synthesized chemical

stimuli. Arrows indicate the direction of water flow. Black bars labelled L-ua (left upstream

antenna), L-da (left downstream antenna), R-ua (right upstream antenna), R-da (right

downstream antenna) indicate passive integrated transponder antennas used to track females.

Dashed lines indicate mesh gates. The centre divider was an aquarium holding a sexually mature
Accepted Article

male. b) Female lake trout increased time spent in the channel treated with male chemical stimuli

but not synthesized prostaglandin F2α (PGF2α). The time spent in the control and experiment

channels before (bc, be) and after (ac, ae) stimulus application were used to calculate an index of

preference for each channel (control = ac/[ac + bc]; experimental = ae/[ae + be]). Wilcoxon

signed-rank tests comparing the control and experimental indices indicated attraction to male-

conditioned water (W = 76, N = 10, P = 0.05), male urine (W = 83, N = 10, P = 0.01), and male

bile (W = 71, N = 9, P = 0.01), but not PGF2α (10-11 M: W = 55, N = 10, P = 0.74; 10-10 M: W =

54, N = 10, P = 0.8). Data are presented as the difference between the indices for the

experimental and the control channels (experimental – control; mean ± se). Points indicate these

values for each individual trial.

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 a) b)
1 0.01
0.05 0.01
0.74
0.6
Accepted Article

0.8

Index of preference
0.2

0 1 2 3 4 5 6

-0.2

-0.6

Male Urine Bile 10-11 10-10

-1 PGF2α

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