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RESEARCH ARTICLE
Male lake char release taurocholic acid as part of a
mating pheromone
Tyler J. Buchinger
1
,KeLi
1,
*, Ugo Bussy
1,‡
, Belinda Huerta
1,§
, Sonam Tamrakar
1
, Nicholas S. Johnson
2
and
Weiming Li
1,¶
ABSTRACT
The evolutionary origins of sexual preferences for chemical signals
remain poorly understood, due, in part, to scant information on the
molecules involved. In the current study, we identified a male
pheromone in lake char (Salvelinus namaycush) to evaluate the
hypothesis that it exploits a non-sexual preference for juvenile odour.
In anadromous char species, the odour of stream-resident juveniles
guides migratory adults into spawning streams. Lake char are also
attracted to juvenile odour but have lost the anadromous phenotype
and spawn on nearshore reefs, where juvenile odour does not persist
long enough to act as a cue for spawning site selection by adults.
Previous behavioural data raised the possibility that males release a
pheromone that includes components of juvenile odour. Using
metabolomics, we found that the most abundant molecule released
by males was also released by juveniles but not females. Tandem
mass spectrometry and nuclear magnetic resonance were used to
identify the molecule as taurocholic acid (TCA), which was previously
implicated as a component of juvenile odour. Additional chemical
analyses revealed that males release TCA at high rates via their urine
during the spawning season. Finally, picomolar concentrations of
TCA attracted pre-spawning and spawning females but not males.
Taken together, our results indicate that male lake char release TCA
as a mating pheromone and support the hypothesis that the
pheromone is a partial match of juvenile odour.
KEY WORDS: Receiver bias, Communication, Chemical signal,
Sensory trap
INTRODUCTION
Some of the most striking exhibits of Earth’s biodiversity are sexual
signals used by animals to gain access to mates. Unsurprisingly,
these traits still captivate evolutionary biologists 150 years after
leading Darwin to his idea of sexual selection (Darwin, 1871). In
recent years, intensive study of calls, colours and displays has
cultivated much theory regarding how preferences for sexual signals
evolve. Classic models emphasize the benefits that signals provide
to the choosing sex (Andersson and Simmons, 2006) whereas
receiver bias models suggest signals exploit pre-existing aspects of
receivers’sensory biology (Ryan and Cummings, 2013). Although
the goal of these models is to describe common trends across the
animal kingdom, studies that provide empirical data have largely
focused on a minority of animals and on traits that are conspicuous
to humans (Coleman, 2009; Cummings and Endler, 2018; Zuk
et al., 2014).
The evolution of sexual preferences remains particularly poorly
understood for chemical signals, due, in part, to limited information
on the molecules involved (Yohe and Brand, 2018). Chemical traits
such as pheromones are the only type of signals likely to be used
across the animal Tree of Life because, among other reasons, many
animals lack vision and hearing (Buchinger and Li, 2023).
Nevertheless, only in insects and a few species of other taxa are
the identities of molecules that act as sexual signals known. A
possible consequence is that studies on chemically based mate
choice have primarily investigated the attributes of signallers (e.g.
nutritional, mating or infection status) that correlate with variation in
their odours and receivers’preferences for them (Johansson and
Jones, 2007), which is a feasible and interesting line of research to
pursue without knowing the identity of the compounds. However,
this relatively narrow focus on signals that benefit receivers by
providing useful information has left receiver bias models little
tested as possible evolutionary mechanisms underlying preferences
for chemical signals (Yohe and Brand, 2018).
Male lake char, Salvelinus namaycush (Walbaum 1792), release a
mating pheromone hypothesized to exploit a non-sexual preference
for juvenile odour (Buchinger et al., 2015, 2017). Odours of juvenile
lake char and its congener Arctic char (Salvelinus alpinus) attract
conspecific adults (Buchinger et al., 2015; Selset and Døving,
1980). In Arctic char, this attraction is a mechanism by which adults
navigate from feeding habitat in the ocean to spawning habitat in
streams, where juveniles reside (Nordeng, 1971, 2009). Lake char,
however, have lost the migratory phenotype (McLennan, 1994; but
see Jones and Ratterman, 2009; Loftus, 1958) and complete their
life cycle in lakes where most populations spawn on nearshore reefs
several months after juveniles from the previous year-class have left
(Deroche, 1969; Martin, 1957). Juvenile odour, although attractive
when fresh (Buchinger et al., 2015; Foster, 1985), is unlikely to
function as a navigational cue for adults because it does not persist at
spawning sites between the time when juveniles emigrate (spring)
and adults return to spawn (autumn; Buchinger et al., 2017).
Interestingly, male lake char arrive at spawning sites before females
(Muir et al., 2012) and release an odour that, like juvenile odour,
attracts males and females (Buchinger et al., 2015). Furthermore,
males do not behaviourally discriminate between male and juvenile
odour and females are attracted to male bile, which contains the
bile acids that are likely components of juvenile odour (Buchinger
et al., 2020; Zhang et al., 2001). These observations raise the
Received 27 September 2023; Accepted 13 December 2023
1
Department of Fisheries and Wildlife, Michigan State University, East Lansing,
MI 48824, USA.
2
US Geological Survey, Great Lakes Science Center, Hammond
Bay Biological Station, Millersburg, MI 49759, USA.
*Present address: Yantai Institute of Coastal Zone Research, Chinese Academy of
Sciences, 264003 Shandong, China.
‡
Present address: Mars Inc., McLean, VA
22101, USA.
§
Present address: Department of Chemistry and Biochemistry,
Southern Connecticut State University, New Haven, CT 06515, USA.
¶
Author for correspondence (liweim@msu.edu)
T.J.B., 0000-0002-4590-341X
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
1
© 2024. Published by The Company of Biologists Ltd
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Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
possibility that the non-sexual attraction to juvenile odour generated
sexual selection on males to release some of the same molecules as
juveniles (i.e. ‘exploit’the non-sexual attraction to juvenile odour;
Christy, 1995).
We sought to identify the male pheromone in lake char to evaluate
the hypothesis that it is a partial match of juvenile odour. Whereas
previous pheromones in fish were identified using natural product
chemistry or screening of commercially available hormones (Li
et al., 2018), our prediction that males release a molecule that is
present in the odour of juveniles but not females alluded to an
alternative approach not yet used to identify fish pheromones:
metabolomics (Kuhlisch and Pohnert, 2015). Metabolomics is the
global analysis of small molecules in which chemical features,
represented by a retention time and mass-to-charge ratio, are
compared across groups (Liu and Locasale, 2017). We profiled
chemical features of the lake char exometabolome, which consists of
the molecules emitted by organisms into the environment and
therefore relevant to chemical communication (Izrayelit et al., 2012;
Viant et al., 2017), to pinpoint any compounds released by males and
juveniles but not females. Using this approach followed by tandem
mass spectrometry (MS), nuclear magnetic resonance (NMR), ultra-
high performance liquid chromatography–MS/MS (UHPLC-MS/
MS) and behavioural assays, we identified taurocholic acid (TCA) –
a bile acid previously implicated as a component of juvenile odour
(Zhang et al., 2001) –as a male mating pheromone in lake char.
MATERIALS AND METHODS
Experimental animals
All experiments were approved by the Michigan State University
Animal Use and Care Committee (Animal Use Form Numbers 08/12-
148-00, 09/15-135-00, PROTO201800064 and PROTO202100198).
Fish were provided by (1) the US Fish and Wildlife Service Sullivan
Creek National Fish Hatchery or (2) the US Geological Survey, Great
Lakes Science Center, Hammond Bay Biological Station (HBBS),
who captured the fish during the spawning season (mid-late October)
from a spawning reef in Lake Huron via hook and line. Details of
source, strain and ages are provided in the respective experiments
below. Experimental animals were stored, sampled and observed at
HBBS. Fish from hatcheries were maintained in tanks separated by
sex and held at approximately 10°C and a 14 h light:10 h dark
photoperiod and then switched to mixed-sex tanks held at 8°C and an
11 h light:13 h dark photoperiod to induce sexual maturation
(spermiation and ovulation; Buchinger et al., 2015). All fish were
held at ambient Lake Huron temperatures and an 11 h light:13 h dark
photoperiod once sexually mature. Fish were given unique tags to pair
individual biological data (sex, length, mass) with experimental results
andtohelptrackwhichindividualswereusedineachexperiment.
Individuals were implanted with a 23 mm passive integrated
transponder tag (PIT tag; Oregon RFID, Portland, OR, USA) via a
small incision in the abdomen while briefly immobilized using 0.08%
(by volume) clove oil or tagged with unique combinations of up to
three streamer tags inserted through the dorsal fin. Adults were fasted
throughout the duration of the experiments. All experiments with live
animals were done at night as lake char are primarily nocturnal during
spawning and, unless otherwise noted, during the autumn (October–
December) spawning season (Muir et al., 2012).
Metabolomic analysis of lake char odours
High resolution-mass spectrometry (HR-MS) on water conditioned
by lake char was used to search for putative pheromone components
released by males and sexually immature juveniles but not females.
Fish used in this experiment were Seneca Lake strain and either age
9–10 years (adults; males: n=6, 70.0±5.9 cm, 3.3±1.21 kg; females:
n=6, 69.7±5.3 cm, 3.17±1.01 kg, means±s.e.m.) or age 4 years
(juveniles; n=15, 42.5±4.2 cm, 0.7±0.21 kg). Char-conditioned
water was collected by placing an individual adult, or 3 juveniles in
tanks with 150 l of flow-through ambient-temperature Lake Huron
water. Unconditioned Lake Huron water was also collected
as a negative control. After a 24 h acclimation of fish to the tank,
the flow was shut off and, after 4 h of odour accumulation, 1 liter of
water was collected and frozen below −20°C. Later, samples were
thawed, and 10 ml was aliquoted and refrozen. The 10 ml
subsamples were then freeze-dried using a CentriVap Cold Trap
with CentriVap Concentrator (Labconco, Kansas City, MO, USA)
and reconstituted in 100 μl of methanol and water (1:1, v:v).
Reconstituted water samples were then subjected to UHPLC
coupled to a Xevo G2-S Q-Tof system (Waters Corporation,
Milford, MA, USA). Metabolites in reconstituted samples were
separated using an ACQUITY C18 BEH UHPLC column
(2.1×100 mm, 1.7 μm particle size; Waters Corporation). The
column temperature was set at 30°C. The mobile phase consisted
of water (A) and acetonitrile (B). The gradient elution was
completed using the following gradient program at a flow rate of
250 µl min
–1
for 10 min: 80% A for 1 min; decreased to 0% A from
1 to 7 min; maintained at 0% A from 7.01 to 9.0 min; back to 80% A
from 9.01 min; and maintained to 10 min for column equilibrium.
To avoid cross-contamination of samples during the analysis, the
needle was washed twice with 80% methanol after each injection.
Carry-over from analyte residues was also reduced by injecting
10 μl methanol as a ‘rinsing solution’on the column after each
sample injection using the elution gradients described above.
MS was performed using the negative electrospray ionization
mode. For the full-scan MS analysis, spectra were recorded in the
range of m/z100–1000. Nitrogen gas was used as both the
desolvation gas (600 l h
–1
) and cone gas (50 l h
–1
); argon was used
as the collision gas at a pressure of 5.3×10
–5
Torr (0.007 Pa). The
source and desolvation temperatures were 100 and 400°C,
respectively; the cone voltage and capillary voltage were set to
30 V and 2.8 kV, respectively. The scan time was set at 0.2 s, with an
interscan delay of 0.5 s. The LockSpray™dual electrospray ion
source with internal references used for these experiments was
leucine enkephalin at a concentration of 100 ng ml
−1
. Lock-mass
calibration data at m/z554.2615 in negative ion mode were acquired
for 1 s every 10 s interval and the flow rate was set at 5 µl min
–1
.For
MS/MS, the voltage settings were switched in a quasi-simultaneous
fashion to produce non-selective collision-induced dissociation, with
collision energies of 5, 10, 20, 30, 40 and 50 eV (Bao et al., 2014).
Kruskal–Wallis rank sum tests followed by pairwise Wilcoxon
rank sum tests were used to compare the magnitudes of the top five
peaks detected in male-conditioned water with the same five peaks
in female, juvenile and control water. No P-value adjustment was
applied in pairwise Wilcoxon rank sum tests as they were used to
explore the data and identify candidate pheromones to be further
evaluated in follow-up analyses.
Finally, TCA concentrations in metabolomics samples were
quantified using an established method of UHPLC-MS/MS (Li et al.,
2015). Reconstituted samples were subjected to UHPLC-MS/MS
(Waters Acquity H-class ultra-performance liquid chromatography
system; Xevo TQ-S triple mass spectrometer, Waters Corporation)
using previously described methods (Li et al., 2015), which include a
comparison with authentic TCA. As above, concentrations of TCA (in
ng ml
−1
) were compared with Kruskal–Wallis rank sum tests
followed by pairwise Wilcoxon rank sum tests with no P-value
adjustments.
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RESEARCH ARTICLE Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
Isolation of TCA from male urine
To unequivocally confirm the identity of TCA as a component of
male odour, we isolated it from male urine and subjected it to NMR.
We focused on urine because previous research implicated it as a
source of male pheromone in lake char (Buchinger et al., 2020) and
we expected urine would be more likely than conditioned water to
yield the amount of molecule necessary for NMR. Urine was
collected during 2014–2016 from sexually mature male lake char
using urinary catheters as previously described (Buchinger et al.,
2020). Briefly, males were anaesthetized using clove oil,
catheterized with 2 mm tubing inserted into the urinary bladder
and secured to the anal, pelvic and dorsal fins, and held in aquaria
(∼200 l) supplied with ambient temperature flow-through Lake
Huron water. Urine (∼11 l) was collected at least once per day into
beakers held on ice, and frozen at <−20°C.
The pooled urine was freeze dried and combined to obtain extract.
Theextractwasthenreconstitutedinmethanoltoremoveinorganic
salts and freeze dried a second time. The final extract (∼500 mg) was
dissolved in waterand methanol (90:10, v:v) and subjected to a reverse
C18 column (house prepared). Fractions (n=50) were eluted using an
increasing methanol:water gradient, screened using UV-activated thin-
layer chromatography (TLC). Similar fractions were combined and the
samples concentrated via freeze drying. Samples were then subjected
to semi-preparative column chromatography (Luna, 250×10 mm,
5 µm) at ambient temperature. The mobile phase consisted of water (A)
and methanol (B). The gradient elution was completed using the
following gradient program at a flow rate of 3 ml min
–1
for 60 min:
90% A for 10 min; decreased to 0% A from 10 to 45 min; and then
maintained at 0% A from 45.01 to 60.0 min. The eluents were
collected by an autosampler (one fraction per minute). Each fraction
was sampled (100 µl) for HR-MS and MS/MS as described above and
NMR. An AVANCE 600 MHz instrument (Agilent, Santa Clara, CA,
USA) was used to conduct the
1
Hand
13
C/DEPT-NMR, two-
dimensional homonuclear (i.e. COSY) and heteronuclear (i.e. HMQC
and HMBC) experiments using previously described parameters (Li
et al., 2021).
TCA release via urine
Urine as a possible route of TCA release was further investigated by
sampling wild spermiated male (n=7, 71.53±7.48 cm, 3.31±1.16 kg)
and ovulated female (n=7, 74.61±3.69 cm, 3.38±0.53 kg) lake char.
First, individual lake char were acclimated in 150 l of ambient
temperature, aerated Lake Huron water for approximately 20 h. The
incoming water was shut off several hours prior to sunset and odour
was allowed to accumulate for 4 h. A 50 ml water sample was spiked
with 10 ng 4-deuterated TCA as an internal standard and frozen at
−20°C. The same individuals were then briefly anaesthetized using
clove oil, catheterized with 2 mm tubing inserted into the urinary
bladder and secured to the anal, pelvic and dorsal fins, and returned
to their tank to recover. Approximately 20 h later, urine was collected
into a 50 ml vial for 4 h. After 4 h, 1 ml samples of urine were spiked
10 ng 4-deuterated TCA and frozen at −80°C until subsequent
analysis using UHPLC-MS/MS (Li et al., 2015). Concentrations (in
ng ml
−1
) of TCA in urine and water were compared between males
and females using Wilcoxon rank sum tests.
TCA release rate
Rates of TCA release were estimated for wild males during
(November; n=12, 74.38±4.79 cm, 3.12±0.66 kg) and outside
(April; n=12, 73.42±5.09 cm, 2.78±0.59 kg) the spawning season.
Males were acclimated in approximately 560 l of ambient Lake
Huron water for at least 20 h. Around sunset, the incoming water
was shut off and 50 ml water was sampled to quantify the baseline
concentration of TCA in the water. After 4 h, a second 50 ml water
sample was collected. A set of negative control samples were also
collected in April using the same procedure but with no fish added
to the tanks (n=6). Sub-samples (10 ml) were freeze dried,
reconstituted in 1 ml methanol, freeze dried again, reconstituted in
100 μl of 50% methanol:water (v:v), and subjected to UHPLC-MS/
MS (Li et al., 2015). The final concentrations (in ng ml
−1
)ofTCA
after 4 h were compared among spawning season samples, non-
spawning season samples and negative control samples using a
Kruskal–Wallis rank sum test followed by a pairwise Wilcoxon test
with a Benjamini–Hochberg adjustment for multiple comparisons.
To estimate release rates, we calculated the difference in TCA
concentration between samples collected at the beginning (which
included TCA released during acclimation) and end (which
included TCA released during acclimation plus TCA released
during the experiment) of odour collection.
Behavioural responses to TCA
Behavioural responses of pre-spawning and spawning male and
female lake char to TCA were evaluated using a pair of identical
two-choice arenas (see Fig. 1 for dimensions). Synthesized TCA
was purchased from Cayman Chemical (www.caymanchem.com;
item no. 16215) and a stock solution prepared in methanol and water
(1:1, v:v). During trials, TCA was mixed into 9 l of Lake Huron
water and applied at 200 ml min
−1
using a peristaltic pump to reach
a concentration of 1 pmol l
−1
when mixed throughout the full width
of the arena. Arenas were built in a flow-through cement flume at the
HBBS and supplied with ambient Lake Huron water. A trial began
30 min after sunset when an individual fish was placed in the arena
to acclimate. After 30 min, fish were observed for 30 min without
any TCA being pumped. TCA was then pumped for a total of
45 min, with a 15 min pre-observation period that allowed the
Velocity=0.012 m s−1
Odour A
1.5 m
Depth=0.4 m
6 m
Flow
Odour B
1.4 m
Observation area
Fig. 1. Behavioural arena used to evaluate responses of adult lake char
(Salvelinus namaycush) to taurocholic acid (TCA). Individuals were
observed for 30 min before and during application of 1 pmol l
−1
TCA into one
channel (e.g. odour A) and a vehicle control into the second channel (e.g.
odour B). Arenas were supplied with Lake Huron water.
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RESEARCH ARTICLE Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
odorant to saturate the activated channel and downstream portion of
the arena and a 30 min observation period during which fish were
observed. All trials were conducted at night in the dark and the time
fish spent in each channel was recorded using infrared cameras. A
human observer recorded the time that fish spent in each channel.
The observer was not blind to the treatments because of staffing
limitations but had no prior expectations as to what responses were
predicted. At the conclusion of a trial, the fish was removed, and
odours flushed for 15 min before starting the next trial. Dye tests
confirmed that 15 min was sufficient to flush the odour from the
flume. The R package lme4 (Bates et al., 2015) was used to
construct a linear mixed effect model with a response variable of
proportion of time spent in the channel activated with TCA
[pTCA=sTCA/(sTCA+sCon); where sTCA is time (s) spent in the
channel treated with TCA and sCon is time (s) spent in the channel
treated with vehicle control], a fixed effect of period ( pre-odour
versus odour), and random effect of fish ID. Estimated marginal
means and 95% confidence intervals were generated using the
emmeans package (https://CRAN.R-project.org/package=emmeans)
and were compared to 0.5, the expected proportion of time
in the activated channel given no response to TCA. Lake char
tested for responses to TCA were Seneca Lake strain age 6 males
(n=25, 59.61±3.49 cm, 2.36±0.38 kg) and age 7 females (n=25,
64.06±3.67 cm, 2.75±0.48 kg).
RESULTS
Most abundant peak in male exometabolome is also present
in that of juveniles but not females
Metabolomics using HR-MS revealed the most abundant peak
(based on means) in male-conditioned water was the fifth most
abundant in juvenile-conditioned water (Fig. 2A). The relative
concentrations of peak 1, which had a retention time of 2.85 min
(Fig. 2B), were similar in male (n=6) versus juvenile (n=6) water
(Wilcoxon rank sum P=0.08), were not different female (n=5) versus
control (Lake Huron; n=5) water (P=0.21), and were lower in female
and control versus male and juvenile water (P<0.05). The second and
fifth most abundant peaks in male-conditioned water were at similar
relative concentrations across all four groups (male, juvenile, female
and control; Kruskal–Wallis P>0.05), whereas the third and fourth
most abundant peaks were detected at higher relative concentrations
in male and juvenile water versus female and control water (P<0.05;
Fig. 2A). Peaks 3 and 4 were at similar relative concentrations in
male versus juvenile water (peak 3 P=0.66, peak 4 P=0.13), though
they tended to be higher in male odour, and in female versus control
water (peak 3 P=0.56, peak 4 P=1). Based upon its m/zof 514.2853
±0.0009 [M−H]
–
(mean±s.e.m.), male peak 1 was putatively
annotated as TCA (Δ=3.77±1.36 ppm).
MS/MS and NMR confirm male peak 1 is TCA
MS/MS confirmed that peak 1 in male-conditioned water and peak 5 in
juvenile-conditioned water were TCA. Electrospray ionization MS
under negative ion mode yielded fragments of 79.9 (−SO
3
–
) and 124.01
(taurine; C
2
H
6
NO
3
S; Fig. 3A), which are characteristic of TCA
(Kaya et al., 2018). UHPLC-MS/MS confirmed that TCA was
at higher concentrations in male- and juvenile-conditioned water
(male 27.39±5.44 nmol l
−1
, juvenile 5.91±4.03 nmol l
−1
)thanin
female-conditioned water (0.06±0.02 nmol l
−1
)ortheLakeHuron
controls (0.02±0.02 nmol l
−1
;P<0.05; see Fig. 3B for concentrations
in ng ml
−1
).
Finally,
1
H and
13
C NMR on the residues of urine extract
confirmed that TCA was a major chemical constituent of male urine
(Table S1,Figs S1–S5).
Males release TCA at high rates via urine during the
spawning season
Spawning male lake char released TCA via urine at high rates during
the spawning season. TCA concentrations were higher in male urine
(28.21±4.48 µmol l
−1
;n=7) than in female urine (1.35±1.01 µmol l
−1
;
0
BA
Peak no.
Male
Juvenile
Time (min)
Female
1:00 2:00 3:00 4:00 5:00
Peak 1
12345
0
50
100
0
50
100
Relative intensity (%)
Relative abundance
100
50
00
500
1000
1500
2000
2500
Male
Female
Juvenile
Control
Fig. 2. The most abundant molecule released by male lake char is also released by juveniles but not females. (A) Metabolomics using high resolution
mass spectrometry (HR-MS) revealed the most abundant compound in male odour (peak 1) was the fifth most abundant in juvenile odour but absent from
female odour and the negative control. Boxplot (median, upper and lower quartiles and 1.5× interquartile range) shows relative peak areas of the top five
compounds detected in water conditioned by males. (B) Representative chromatographs showing peak 1 in water conditioned with male and juvenile but not
female lake char. n=6 for males and females, n=5 for juveniles and control.
4
RESEARCH ARTICLE Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
P=0.001; n=7), consistent with the higher concentrations of TCA
measured in water conditioned with the same individual males
and females 24 h pre-catheterization (male 18.96±6.10 nmol l
−1
,
female 1.04±0.42 nmol l
−1
;P=0.004; Fig. 4). TCA concentrations
were higher in water conditioned by males during versus
outside the spawning season (spawning 7.18±3.09 nmol l
−1
,
n=12; non-spawning 0.07±0.01 nmol l
−1
,n=12; P<0.001; Fig. 5),
though water conditioned by males at either time point had higher
TCA concentrations than detected in the negative control (Lake
Huron) samples (0.02±0.001 nmol l
−1
;n=6; P<0.05). Paired
samples collected at the start (0 h) and end of the odour
accumulation period (4 h) to were used estimate TCA release rates
at 76.58±22.66 µg kg
−1
h
−1
(0.15±0.04 µmol kg
−1
h
−1
) for males
during the spawning season (n=12) and 1.38±0.23 µg kg
−1
h
−1
(0.003±0.001 µmol kg
−1
h
−1
) for males outside the spawning
season (n=11; see Fig. 5 for release rates unadjusted by body mass).
One non-spawning male was excluded from the release rate estimate
because its measured TCA concentrations decreased between the
0 h and 4 h sampling periods.
TCA attracts females but not males
In two-channel flumes, pre-spawning and spawning females but not
males spent proportionally more time in the channel activated with
1 pmol l
−1
TCA than in the channel treated with the vehicle (50%
methanol; Fig. 6; 95% confidence interval, CI >0.5, the predicted
neutral response). The proportion of time spent in the treatment
channel during the pre-odour (negative control) period was not
different from 0.5 for any group ( pre-spawning male 95% CI:
0.31–0.61; n=12, pre-spawning female 95% CI: 0.31–0.60; n=16,
spawning male 95% CI: 0.26–0.69; n=11, spawning female 95%
CI: 0.33–0.8; n=11). The average time spent in a channel during the
30 min observation periods ranged from 58.73±27.35 s (control
channel during odour period for spawning females) to 273.5±62.1 s
(control channel during pre-odour period for pre-spawning males).
Two trials were excluded from further analysis because the
individual did not enter either channel during either the pre-odour
or odour periods.
DISCUSSION
Our results indicate that male lake char release a component of
juvenile odour as a mating pheromone. Previous studies have shown
that odorants in juvenile faeces may attract adult lake char
(Buchinger et al., 2017; Foster, 1985) and closely related Arctic
char (Selset and Døving, 1980) to spawning habitat. In the current
study, we found that males, but not females, released the bile acid
TCA, a common digestive metabolite implicated as a component of
juvenile odour (Zhang et al., 2001; Zhang and Hara, 2009; present
results). Whereas juveniles release TCA via faeces (Zhang et al.,
2001), the primary route of bile acid excretion under normal
physiological conditions, males released TCA via urine, which
usually has low concentrations of bile acids (Sato and Suzuki, 2001)
and is an alternative route of bile acid elimination used (in
mammals) during pathological conditions such as cholestasis
(Krones et al., 2018). Males released TCA at high rates but only
during the spawning season. In laboratory assays, picomolar
concentrations of TCA attracted pre-spawning and spawning
females but not males. We suggest the non-sexual role of TCA as
part of juvenile odour preceded its role as a male pheromone
because (1) non-sexual release of TCA via faeces is widespread in
vertebrates (Bogevik et al., 2009; Hagey et al., 2010b; Schmucker
et al., 2020; Velez et al., 2009; Yeh et al., 2012) and likely an
ancestral trait and (2) previous evidence suggests ancestral char use
(or used) the odour of juvenile faeces to navigate to spawning sites
(Buchinger et al., 2017; Foster, 1985; Nordeng, 1971; Selset and
Døving, 1980; Zhang et al., 2001). However, phylogenetic
comparisons are needed to further evaluate whether attraction to
TCA preceded male signalling with TCA. Nevertheless, our results
implicate TCA as a behaviourally active component of juvenile
odour and the male mating pheromone, and are consistent with the
hypothesis that male signalling with TCA exploits a non-sexual
attraction to juvenile odorant.
The few fish pheromones identified using chemistry-driven
approaches (Li et al., 2018) challenge conventional models of
pheromone evolution that emphasize benefits to receivers
(Wisenden, 2015). Many known pheromones in model species
100
00
5
10
15
20
25
50
B
m/z
Male Female Juvenile Control
112.9841
514.2778
124.0108
Relative intensity (%)
TCA concentration (ng ml−1)
Juvenile
A
0
50
100
514.2874
106.9817
79.9569
79.9569
80.9609 124.0048
80
[M−H]
Male
120 160 200 240 280 320 360 400 440 480 520
75 125 175 225 275 325 375 425 475 525
HO
OH
OH
OH
O
S
O
O
C2H6NO3S•
O3S•
124.01
79.9
N
H
Fig. 3. Peak 1 is taurocholic acid (TCA). (A) Representative tandem MS (MS/MS) spectra of male- and juvenile-conditioned water showing characteristic
ion fragments of TCA (Kaya et al., 2018). (B) Concentrations of TCA in samples used for metabolomics as determined using a previously described method
of ultra-high performance liquid chromatography (UHPLC)-MS/MS (Li et al., 2015). n=6 for males and females, n=5 for juvenile and control.
5
RESEARCH ARTICLE Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
such as goldfish are sex steroids and prostaglandins that were
commercially available for testing and predicted to act as
pheromones because of their roles as hormones that leak into the
environment during specific reproductive phases (Stacey, 2015).
This biology-driven approach has provided a wealth of information
on pheromone function and evolution but risks a bias towards
molecules that benefit receivers, as leaked hormones provide
information directly related to the reproductive status of potential
mates. Indeed, two of the three pheromones previously identified
using the alternative approach of isolating molecules from natural
odours are non-hormonal compounds (amino acid: Yambe et al.,
2006; bile acid: Li et al., 2002) with less obvious links to the
reproductive status of the releaser (Buchinger et al., 2014; Li et al.,
2018). Our approach leveraged metabolomics as well as natural
product chemistry but unveiled a second bile acid mating
pheromone. Unlike sex hormones (Stacey, 2015), bile acids are
generally not expected to act as mating pheromones because they are
primarily involved in digestion of fats, not reproduction (Buchinger
et al., 2014). However, in some cases, diet-related differences in bile
acid release may indicate reproductive status (Ashouri et al., 2023).
Furthermore, adult lake char feed little during the spawning season
(Vinson et al., 2021) and the release of large amounts of TCA may
require large energy reserves and therefore reflect male quality.
Regardless, we expect that additional chemistry-driven studies may
continue to reveal unexpected identities of mating pheromones in
fish (Li et al., 2018).
The male pheromone in lake char appears to consist of multiple
components with sex-specific effects (Buchinger et al., 2015).
Pheromones are often mixtures of multiple molecules (Wyatt, 2014)
and previous behavioural evidence that females, but not males,
discriminate between male and juvenile odour indicates the male
lake char pheromone is no exception; males appear to respond to
components released by males and juveniles, whereas females
respond to components released by males and juveniles plus other
components released only by males (Buchinger et al., 2015). In the
current study, we searched for and found a component of juvenile
odour (TCA) that was released by males at high rates during the
spawning season and attracted females. Our experiments did not test
the relative role of TCA as a part of a mixture but did show that TCA
was the most abundant peak in male-conditioned water.
Furthermore, the female responses to TCA we observed were
nearly identical to responses to natural male odorant previously
observed in a comparable assay (when adjusted for different trial
durations; Buchinger et al., 2015). However, TCA at 1 pmol l
−1
did
not attract males, though natural male and juvenile odorants both
attract males (Buchinger et al., 2015, 2017) and consist, in part, of
TCA (Zhang et al., 2001; present results). Additional components,
either alone or as a mixture with TCA, may mediate male responses
to male and juvenile odours. Alternatively, males may have higher
response thresholds to TCA than females, though, to our
knowledge, no data exist on potential sex differences in lake char
olfactory responses (but see Ghosal and Sorensen, 2016).
Interestingly, sea lamprey (Petromyzon marinus) also show sex-
specific responses to components of a male pheromone that attracts
both sexes (Scott et al., 2019). We postulate that males and females
show different responses to chemical constituents of the male
pheromone because it plays different ecological roles for each sex
(e.g. mate search versus male–male competition; see Buchinger
et al., 2015, for more discussion on male responses to male
pheromones). More research is needed to better understand the role
14
A
B
12
10
8
6
Water TCA (ng ml−1)
Urine TCA (µg ml−1)
4
2
0
0
5
10
Male Female
15
20
25
Fig. 4. Male lake char release TCA via urine. (A) Concentrations of TCA in
water conditioned with male and female lake char. (B) Concentrations of
TCA in urine collected from male and female lake char. Urine was collected
via catheter from spawning males (n=7) and females (n=7) 24 h after holding
water was sampled from the same individuals.
700
600
500
400
300
200
Spawning
male
Non-spawning
male
100
0
Rate of TCA release (µg h−1)
Fig. 5. Male lake char release TCA at high rates during the spawning
season. Release rates of TCA were estimated using paired samples
collected at the start (0 h) and end of the odour accumulation period (4 h).
Males were sampled during the spawning season (autumn; n=12) and
outside the spawning season (spring; n=11). TCA was detected a low
concentrations (0.01±0.001 ng ml
−1
) in the negative control (Lake Huron;
n=6) samples. For reference, release rates in molar concentrations were
0.53±0.18 µmol h
−1
for spawning males and 0.008±0.001 µmol h
−1
for
non-spawning males.
6
RESEARCH ARTICLE Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
of TCA as a component of a male pheromone that influences the
behaviour of both males and females.
In conclusion, our study provides evidence that male lake char
release TCA as a mating pheromone that exploits a non-sexual
preference for juvenile odour. Our chemistry-driven approach
implicated an evolutionary mechanism inconsistent with
conventional models of mating pheromones in fish (Stacey, 2015;
Wisenden, 2015) and with little empirical support in even the well-
characterized pheromones of insects (Stökl and Steiger, 2017).
Hypothesis-driven metabolomics proved to be a powerful approach
that may accelerate and enrich research on pheromone
communication in fish and other taxa for which identification of
underlying chemical traits is often logistically prohibitive. As TCA
is a common bile acid produced by many animals (Hagey et al.,
2010a; Hofmann et al., 2010), future work might also evaluate
whether and how the male pheromone in lake char is species
specific, which is an often noted, but not required, characteristic of
pheromones (Buchinger and Li, 2020). Notably, both the current
study and previous work support suggestions that spawning lake
char also use cues detected by other sensory modalities (e.g.
hearing: Johnson et al., 2018) as females spent only about 10% of
the trial in the channel treated with natural male odorant (Buchinger
et al., 2015) or TCA (current study). Other pheromone components
and sensory modalities may allow species-specific communication
despite TCA being a common metabolite. Lastly, a better
understanding of pheromone communication in lake char may
inform management of both native and invasive populations
(Hansen et al., 2019).
Acknowledgements
We thank the US Fish and Wildlife Service Sullivan Creek and Iron River National
Fish Hatcheries, the Michigan Department of Natural Resources Marquette State
Fish Hatchery, and the Ontario Ministry of Natural Resources Chatsworth Fish
Culture Station for supporting this research by providing lake char. Thanks to Carrie
Baker, Luke Baker, Kenneth Bennett, Tyler Bruning, Skye Fissette, Elizabeth
Herbst, Jacob Kimmel, Michaela Kratofil, Melissa Pomranke, Ryan Pokorzynski,
Joseph Rabaey, Trisha Searcy, Margaret Spens and Riley Waterman for assisting
with data collection. Tom Binder, Skye Fissette, Anne Scott and two anonymous
reviewers provided useful comments on the manuscript. We also thank the staff of
the Mass Spectrometry and Metabolomics Core Facilityof Michigan State University
for technical support. Any use of trade, product, or firm name is for descriptive
purposes only and does not imply endorsement by the US Government.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: T.J.B., N.S.J., W.L.; Formal analysis: T.J.B., K.L., U.B., B.H.,
S.T.; Investigation: T.J.B., K.L., U.B., B.H., S.T.; Resources: N.S.J., W.L.; Writing -
original draft: T.J.B.; Writing - review & editing: K.L., U.B., B.H., S.T., N.S.J., W.L.;
Supervision: N.S.J., W.L.; Project administration: N.S.J., W.L.; Funding acquisition:
T.J.B., K.L., N.S.J., W.L.
Funding
This work was supported by the Great Lakes Fishery Trust [2016.1631] and Great
Lakes Fishery Commission [2011_JOH_4415]. Open Access funding provided by
Michigan State University. Deposited in PMC for immediate release.
Data availability
Data and code are available from the Dryad digital repository (Buchinger et al.,
2024): doi:10.5061/dryad.8931zcrvk
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RESEARCH ARTICLE Journal of Experimental Biology (2024) 227, jeb246801. doi:10.1242/jeb.246801
Journal of Experimental Biology
