Behavioral evidence of olfactory imprinting during embryonic and larval stages in lake sturgeon

Abstract

Many migratory fishes are thought to navigate to natal streams using olfactory cues learned during early life stages. However, direct evidence for early-life olfactory imprinting is largely limited to Pacific salmon, and other species suspected to imprint show life history traits and reproductive strategies that raise uncertainty about the generality of the salmonid-based conceptual model of olfactory imprinting in fishes. Here, we studied early-life olfactory imprinting in lake sturgeon (Acipenser fulvescens), which have a life cycle notably different from Pacific salmon, but are nonetheless hypothesized to home via similar mechanisms. We tested one critical prediction of the hypothesis that early-life olfactory imprinting guides natal homing in lake sturgeon: that exposure to odorants during early-life stages results in increased activity when exposed to those odorants later in life. Lake sturgeon were exposed to artificial odorants (phenethyl alcohol and morpholine) during specific developmental windows and durations (limited to the egg, free-embryo, exogenous feeding larvae and juvenile stages), and later tested as juveniles for behavioral responses to the odorants that were demonstrative of olfactory memory. Experiments revealed that lake sturgeon reared in stream water mixed with artificial odorants for as little as 7 days responded to the odorants in behavioral assays over 50 days after the initial exposure, specifically implicating the free-embryo and larval stages as critical imprinting periods. Our study provides evidence for olfactory imprinting in a non-salmonid fish species, and supports further consideration of conservation tactics such as stream-side rearing facilities that are designed to encourage olfactory imprinting to targeted streams during early life stages. Continued research on lake sturgeon can contribute to a model of olfactory imprinting that is more generalizable across diverse fish species and will inform conservation actions for one of the world's most imperiled fish taxonomic groups.

Full text

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Volume 11 • 2023 10.1093/conphys/coad045
Research article
Behavioral evidence of olfactory imprinting
during embryonic and larval stages in lake
sturgeon
Jacob G. Kimmel1,Tyler J. Buchinger1, Douglas L. Larson1,Edward A. Baker2,Troy G. Zorn2,
Kim T. Scribner1, 3 and Weiming Li1,*
1Department of Fisheries and Wildlife, Michigan State University, 480 Wilson Road, East Lansing MI 48824, USA
2Michigan Department of Natural Resources, Marquette Fisheries Research Station, 484 Cherry Creek Road, Marquette, Michigan, 49855, USA
3Department of Integrative Biology, Michigan State University, 288 Farm Lane, East Lansing MI 48824, USA
*Corresponding author: Department of Fisheries and Wildlife, Michigan State University, 480 Wilson Road, East Lansing MI 48824, USA.
Email: Liweim@msu.edu
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Many migratory shes are thought to navigate to natal streams using olfactory cues learned during early life stages. However,
direct evidence for early-life olfactory imprinting is largely limited to Pacic salmon, and other species suspected to imprint
show life history traits and reproductive strategies that raise uncertainty about the generality of the salmonid-based con-
ceptual model of olfactory imprinting in shes. Here, we studied early-life olfactory imprinting in lake sturgeon (Acipenser
fulvescens), which have a life cycle notably dierent from Pacic salmon, but are nonetheless hypothesized to home via similar
mechanisms. We tested one critical prediction of the hypothesisthat early-life olfactor y imprinting guides natal homing in lake
sturgeon: that exposure to odorants during early-life stages results in increased activity when exposed to those odorants later
in life. Lake sturgeon were exposed to articial odorants (phenethyl alcohol and morpholine) during specic developmental
windows and durations (limited to the egg, free-embryo, exogenous feeding larvae and juvenile stages), and later tested
as juveniles for behavioral responses to the odorants that were demonstrative of olfactory memory. Experiments revealed
that lake sturgeon reared in stream water mixed with articial odorants for as little as 7 days responded to the odorants in
behavioral assays over 50 days after the initial exposure, specically implicating the free-embryo and larval stages as critical
imprinting periods. Our study provides evidence for olfactory imprinting in a non-salmonid sh species, and supports further
consideration of conservation tactics such as stream-side rearing facilities that are designed to encourage olfactory imprinting
to targeted streams during early life stages. Continued research on lake sturgeon can contribute to a model of olfactory
imprinting that is more generalizable across diverse sh species and will inform conservation actions for one of the world’s
most imperiled sh taxonomic groups.
Key words: odorants, lake sturgeon, imprinting, behavioral response
Editor: Dr. Steven J. Cooke
Received 4 November 2022; Revised 30 March 2023; Editorial Decision 11 May 2023; Accepted 14 June 2022
Cite as: Kimmel JG, Buchinger TJ, Larson DL, Baker EA, Zorn TG, Scribner KT, Li W (2023) Behavioral evidence of olfactory imprinting during
embryonic and larval stages in lake sturgeon.Conser v Physiol 11(1): coad045; doi:10.1093/conphys/coad045.
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Research article Conservation Physiology • Volume 11 2023
Introduction
Many fish species migrate from feeding grounds to spawning
locations (Lucas and Baras, 2001). The timing and destination
of spawning migrations have implications on the rate of early
development and offspring survival (Reznick et al., 2006;
Forsythe et al., 2012), and therefore are a mechanism by
which adults provide indirect benefits to offspring (Leggett,
1977;Jørgensen et al., 2008). Survival benefits conferred to
offspring based on optimal spawning choices by parents select
for repeated spawning at the optimal location, or spawning
site fidelity, which can lead to localized adaptions and geneti-
cally distinct populations at different spawning sites (Leggett,
1977). In contrast, interbreeding caused by adults straying
into other spawning sites may lead to outbreeding depression,
which is the reduction in fitness because of the breakdown of
coadapted genotypes that are adapted to species and (largely
natal) environments (Edmands, 2007). Understanding the
mechanisms guiding natal site homing is important for the
management of migratory species, and studies have shown
that straying from natal sites may occur more often by stocked
individuals (Quinn, 1993).
Many fish species are hypothesized to use olfactory cues
to home to natal habitat for spawning (Bett and Hinch,
2016). Natal homing via olfactory cues is well-studied in
Pacific salmon (Oncorhynchus sp), which imprint to stream-
specific odors during early life stages, especially the period
of parr-smolt transformation, and then follow these odors at
sexual maturity when navigating to spawning sites (Dittman
and Quinn, 1996). In salmon and other species, research on
olfactory imprinting informs conservation efforts because,
for example, the findings support development of artificial
propagation programs that encourage homing to targeted
locations (Dittman et al., 2015). Despite extensive research on
olfactory imprinting in Pacific salmon and relevance of olfac-
tory imprinting to management of various species, evidence
for olfactory imprinting by non-salmonid species is largely
indirect, such as observed homing to spawning sites or devel-
opment of olfactory structures during suspected imprinting
periods (Horrall, 1981;Cathcart, 2021).
The established model of olfactory imprinting in Pacific
salmon is unlikely representative of many fishes (Bett and
Hinch, 2016). Most research on olfactory imprinting to the
odor of natal habitats in Pacific salmon has focused on coho
salmon (O. kisutch;Bett and Hinch, 2016), which spend
about a year in home streams after hatching, and while
undergoing the metamorphosis-like transition of smolting
(Bishop et al., 2006;Björnsson et al., 2012), move to the ocean
to feed for about 1.5 years before returning to natal streams
to spawn and die. However, many fish, including some species
of Pacific salmon, differ from coho salmon in life history traits
potentially related to olfactory imprinting, such as duration of
pre-smolting occupancy of freshwater habitats. Species such
as pink salmon (O. gorbuscha) and walleye (Sander vitreus)
leave their natal habitats soon after hatching and would need
to imprint much earlier than coho salmon (Horrall, 1981;
Bett et al., 2016). Likewise, most fish develop directly from
embryos to juveniles to adults rather than undergoing any
type of metamorphosis associated with major changes in
levels of thyroid hormones (Rousseau and Dufour, 2012) that
mediate imprinting in Pacific salmon (Dittman and Quinn,
1996;Lema and Nevitt, 2004). Examples of other life history
traits likely relevant to the role of olfactory imprinting in
natal homing, and that differ among species include, the
habitats in which spawning occurs (e.g. lake vs streams),
the age at which adults return to spawn, and whether the
species is semelparous (dies after single spawning season)
or iteroparous (spawns repeatedly across years). Research is
needed to test whether and how olfactory imprinting might
guide natal homing in fishes with life histories different from
Pacific salmon (Bett and Hinch, 2016).
Lake sturgeon (Acipenser fulvescens) have a life cycle
notably different from Pacific salmon (Peterson et al., 2007),
but are nonetheless hypothesized to home to natal streams
to spawn based on a similar process of olfactory imprint-
ing (Holtgren et al., 2007). Age-0 lake sturgeon hatch 8–
14 days post-fertilization (dpf), begin exogenous feeding 13–
19 days after hatching, and move from the river into lakes
after approximately four months (Holtgren and Auer, 2004;
Benson et al., 2005). Males feed and grow in lakes for 12–
20 years and females 14–33 years before becoming sexually
mature (Bruch and Binkowski, 2002;Thiem et al., 2013;
Dammerman et al., 2019). Lake sturgeon movements and
habitat occupation prior to sexual maturity or outside of the
reproductive season vary among individuals and populations,
and include year-around residence in the river, residence in a
lake near the natal river and migrations across large portions
of a lake (Kessel et al., 2018;Scribner et al., 2022). Once
mature, males migrate into streams to spawn every 1–3 years
and females every 4–9 years (Peterson et al., 2007;Forsythe et
al., 2012). Like Pacific Salmon, lake sturgeon show spawning
site fidelity, inferred based on high levels of genetic differenti-
ation among populations (DeHaan et al., 2006;Welsh et al.,
2008;Homola et al., 2012;Donofrio et al., 2018;Scribner
et al., 2022). Observations of spawning site fidelity in lake
sturgeon and high straying rates in shortnose sturgeon (A.
brevirostrum) reared in water from sources other than the
river in which they were stocked (Smith et al., 2002) have
led to the hypothesis that olfactory imprinting guides natal
homing in lake sturgeon (Holtgren et al., 2007).
A better understanding of olfactory imprinting is needed
to support efforts to restore self-sustaining and genetically
diverse populations of lake sturgeon. Once among the most
abundant fishes in the Laurentian Great Lakes, Mississippi
River and Hudson Bay drainages (Scott and Crossman, 1973;
Haxton et al., 2014), lake sturgeon populations in most
areas are now reduced to less than 1% of their historical
numbers (Hay-Chmielewski and Whelan, 1997), and are
considered extirpated, endangered, threatened, or of special
concern in much of their range (Bruch et al., 2016). Together
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Conservation Physiology • Volume 11 2023 Research article
with habitat improvements and fishery restrictions (Welsh,
2004), current efforts to restore lake sturgeon rely heavily
on artificial propagation (Bruch et al., 2016). In particular,
lake sturgeon hatchery programs increasingly employ stream-
side rearing facilities in an effort to imprint young sturgeon
to targeted streams and to encourage subsequent homing by
spawning adults (Holtgren et al., 2007). Although tactics
intended to encourage natal homing via imprinted stream
odors are currently in use, whether and when age-0 lake
sturgeon imprint to odors remains unknown.
In this study, we examined one essential facet of the
hypothesis that olfactory imprinting guides natal homing
in lake sturgeon: specifically, the ability to form olfactory
memory (i.e. store and later retrieve olfactory information;
Zlotnik and Vansintjan, 2019) and during early life stages
(Hino et al., 2009). Age-0 lake sturgeon leave their natal
stream early in life (∼4 months; Holtgren and Auer, 2004;
Benson et al., 2005) and undergo rapid forebrain develop-
ment in key olfactory information centers during the transi-
tion from free-embryos to exogenously feeding larvae (Zhang
and Dang, 2014). Studies in Russian sturgeon (Acipenser
gueldenstaedti) have also provided evidence for olfactory
imprinting during early development, finding elevated thyroid
hormone levels prior to exogenous feeding (Boiko et al.,
2004) and demonstrating learned responses to morpholine
in larval fish with elevated thyroid hormones (Boiko and
Grigor’yan, 2002). Therefore, we predicted that imprinting—
defined here as learning that occurs during a developmentally
sensitive period and results in responses that persist outside
of that period (Immelmann, 1975) in lake sturgeon occurs
during the free-embryo (12–18 dpf) and larval stages (19–49
dpf). Although our overarching hypothesis pertains to odor-
mediated homing by adult lake sturgeon, we tested behavioral
responses of juveniles to odorants experienced during early
life because raising individuals to adulthood (10–30 years)
was not feasible. Research on Pacific salmon indicates young-
of-year individuals display attraction to their natal river water
(Bodznick, 1978), and this response has been a useful proxy
for responses of adults to imprinted odors (Dittman et al.,
2015). We exposed age-0 lake sturgeon to artificial odorants
during specific developmental stages and later tested their
behavioral responses to the odorants to determine whether
they were recognized during later life stages. Our experiments
provided rare evidence for olfactory imprinting in a non-
salmonid, identified periods during which imprinting likely
occurs in lake sturgeon, and will inform restoration efforts
that increasingly rely on hatchery stocking to rebuild or
reintroduce populations in specific streams.
Methods
Experimental animals
Lake sturgeon used in experiments were reared from eggs fer-
tilized at the Black Lake Sturgeon Rearing Facility in Onaway,
MI, USA, which operates as a f low-through streamside rear-
ing facility (SRF) using water supplied directly from the Upper
Black River at ambient temperature (ranging from 8◦Cto
28◦C over the course of the experiment). Eggs and sperm
were sampled from spawning lake sturgeon in the Upper
Black River on May 4, 2021. Eggs were fertilized within
8 hrs following standardized hatchery procedures (Crossman
et al., 2011;Bauman et al., 2015). Offspring from one male
and one female were used in the experiments. The use of
full siblings was expected to reduce variation due to additive
genetic effects (Dammerman et al., 2015,2020). Experimental
animals were used with approval from the Michigan State
University Animal Use and Care Committee (Animal Use
Form # PROTO202000023/AMEND202100062).
Fish were raised in 68 L tanks in a flow-through system
with 50 micron filtered stream water from the Black River.
Within the 68 L tanks, fertilized eggs were held in McDonald
hatching jars (Pentair, Apopka, FL) (held at a density of less
than 200 eggs). Hatched fish were held in 3 L aquaria with bio
ball filters (CBB1-S; Pentair, Apopka, FL) to simulate natural
stream substrate (held at a density of less than 100 individ-
uals), and exogenous feeding fish in 3 L aquaria without
bio ball filters (held at a density that ranged from 25 to 50
individuals). Fish were moved to the larger 68 L tanks for the
juvenile stage (held at a density of less than 50 individuals).
While tank turnover rates varied by tank type, on average
tanks experienced 10 turnovers per hour, and inflow rates
were increased during periods of high temperatures during
the summer months to reach closer to 40 turnovers per hour.
Throughout our experiments, fish were fed a diet of brine
shrimp (Artemia spp.) from 19 to 49 dpf and bloodworms
(Diptera: Chironomidae) after 49 dpf.
Exposure to experimental imprinting
odorants
Fish were exposed to two experimental odorants that were
continually mixed in 50 micron filtered river in the hatch-
ery, phenethyl alcohol (PEA) at 1.04 x 10−7M and mor-
pholine at 9.9 x 10−11 M concentrations. Odorants were
purchased from Sigma-Aldrich Co., Saint Louis, MO, USA
at ≥99% purity. Odorants and concentrations were selected
based upon olfactory imprinting studies in Pacific salmon
(Bett and Hinch, 2016). PEA and morpholine are potent
odorants for fish (Scholz et al., 1976), allowing control of
the exact concentrations and periods during which odorants
were experienced without potentially confounding effects of
background odorants. Odorant exposure occurred during
four early developmental stages: (1) fertilized egg (0 dpf),
(2) hatched free-embryos (12 dpf, ∼9 mm total length), (3)
exogenously feeding larvae (19 dpf, ∼20 mm total length)—
when individuals began feeding on brine shrimp (Artemia
spp.), and (4) juveniles (49 dpf, ∼45 mm total length)—when
individuals began feeding on blood worms (Diptera: Chirono-
midae) (Fig. 1). Lake sturgeon exposure to odorants was orga-
nized into ten unique combinations of developmental stage
treatment to determine whether olfactory imprinting occurred
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Research article Conservation Physiology • Volume 11 2023
Figure 1: Timing and duration of the four developmental stages
(Egg, Free-Embryo [FE], Exogenous Feeding Larvae [Larvae], and
Juvenile [Juv.]) and ten experimental odorant exposure treatments.
Start time for each developmental stage refers to dpf and stage
duration refers to the total length (days) of each developmental
stage. Exposure stages associated with each treatment are indicated
in white (developmental stages without odorant exposure) and grey
(developmental stages with odorant exposure). Exposure length was
measuredindays.
during a specific stage or combination of stages. Individuals
in each treatment were raised in three replicate tanks, but
some replicate tanks were lost due to mortality. Overall,
mortality was low (less than 5% of fish) during this study,
with small increases in fish mortality during high temperature
events, which were unrelated to the timing of odor appli-
cation. Individuals and replicates in one (control) treatment
were never exposed to experimental odorants. Individuals
and replicates in another treatment were exposed during all
stages. Individuals and replicates in four treatments were
exposed during a single developmental stage, and individuals
and replicates in four treatments were exposed during two to
three consecutive developmental stages including either the
egg, free embryo (FE), exogenous feeding larvae, or juvenile
(juv.) stage.
The developmental stages selected represent four distinct
periods of development, behavior and location/habitats lake
sturgeon occupy in natural stream environments when olfac-
tory imprinting may occur. The beginning and end of each
stage were determined by critical thermal units and physiol-
ogy (Eckes et al., 2015). The egg stage begins immediately
with fertilization in the water column after which eggs adhere
to the stream substrate. The FE stage begins at hatch and
is the period when lake sturgeon burrow into the substrate
to avoid predators and consume their yolk-sac (Kempinger,
1988;Detlaff et al., 1993). The exogenous feeding larvae stage
represents the period when fish have depleted their yolk-sac,
begun feeding from the external environment and emerged
from the gravel to drift downstream in river currents (Auer
and Baker, 2002). Individual larvae typically drift for one to
two days and the juvenile stage for our experiment occurred
thirty days after larval drift, when lake sturgeon forage for
food in juvenile habitat of their natal river system.
Odorant stock solutions were mixed daily in3Lofhatch-
ery water to create our odor mixture; a 10 ml stock solution
was used for PEA and a 1 ml stock solution was used for
morpholine. We calculated the concentrations for the stock
solutions based on the final volume of the odor mixture and
this mixture was applied to our system water to establish the
proper concentration of each odorant in our experimental
tanks. A peristaltic pump (model: BT100S, Golander, Duluth,
GA) was used to pump the odor mixture into an 88 L head
tank supplying water to tanks receiving the experimental
odorants. Rhodamine dye (Sigma-Aldrich Co., Saint-Louis,
MO, USA) was pumped into the head tank to reach a concen-
tration of 100 ppb and measured using a hand-held DataBank
datalogger and Cyclops-7 Optical Rhodamine Dye Tracer
(Turner Designs, Sunnyvale, CA) to visually validate mixing of
odorants in the head tank and to validate the even distribution
of odorants supplied to all tanks. We recorded the time of
odorant mixture replacement and the volume of odorant
mixture remaining each day to track daily odorant concen-
trations across all tanks. Using the volume and duration of
odorant mixture pumped each day, we calculated an average
concentration of 9.71 ±0.22 x 10−8M (mean ±standard
error [SE]) for PEA and 9.24 ±0.21 x 10−11 M (mean ±SE)
for morpholine over the duration of the experiment.
Behavior experiments
Juvenile lake sturgeon swimming and activity behaviors were
observed in response to PEA and morpholine as a test of
olfactory memory of the artificial natal odorants. Twenty
individuals were observed from each treatment, using an
equal number of individuals for each replicate. Trials took
place in a cylindrical tank with a 1552 cm2(44.4 cm diameter)
base filled with 3 L (or a depth of 1.9 cm) of groundwa-
ter from the facility (Supplementary Fig. S1). Four identical
arenas were used to measure behaviors based on multiple
estimated movement parameters, allowing for multiple trials
to be run concurrently. Prior to experiments we measured
behavioral responses to food odors (bloodworms) and spring
water (negative control) to verify our behavioral assay could
detect changes in behavior after exposure to a specific odorant
and not simply any substance. For each trial, one individual
fish was removed from its housing tank and a photo was taken
for body length measurements. Fish were then acclimated to
the enclosure for seven and a half minutes which, based on
preliminary observations, was the time when total activity
reduced to normal levels for most individuals. Videos were
recorded for five minutes after acclimation to measure pre-
odor behaviors. Odorant solutions were created to reach the
desired concentrations of 1.04 x 10−7M for PEA and 9.90
x10
−11 M for morpholine in the behavioral arena. A volume
of 100 ml of odorant stock solution was added using two
50 ml syringes; observers were instructed to add the odors
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Conservation Physiology • Volume 11 2023 Research article
to the water using a zig-zag pattern evenly across the surface
in the behavioral arena over a five second period. Applying
the odors across the surface using two syringes resulted in
even application and mixing of the odorant throughout the
arena, as confirmed using dye tests. One minute after the
initial addition of odorants, another five-minute video was
recorded to measure behaviors post-odor application. Fish
were then removed from the enclosures, and enclosures were
thoroughly rinsed with groundwater before the next trial
began. To prevent outside stimuli from affecting fish behav-
iors, fish were observed in the evening outside of working
hatchery hours and in the dark under red lighting. Length was
measured for each fish with the ImageJ software (National
Institutes of Health, Bethesda, MD, U.S.A.; http://rsbweb.
nih.gov/ij/). Videos were analyzed using Loligo v.4.0 track-
ing software (Loligo Systems, Viborg, Denmark; https://www.
loligosystems.com/software), which recorded average velocity
(cm/s), average acceleration (cm/s2), average deacceleration
(cm/s2), time active (s), time active (%), time inactive (s), time
inactive (%) and total distance traveled (cm).
Statistical analyses
All analyses were conducted using R version 4.1.2 (R Core
Team, 2021). To identify relationships between recorded
behavioral movement metrics and to select an informative
metric to use in our analysis, we calculated correlations
between response variables using the corrplot package (v0.92;
Wei and Simko, 2021). Using the absolute value of Pearson
correlation coefficients, we found strong pairwise correlations
(|r| ≥0.89) between average velocity, average acceleration,
average deacceleration and total distance traveled variables
(Supplementary Fig. S2). We also found strong correlations
(|r| ≥0.93) between time active and time inactive measures.
There was a moderate correlation (|r| >0.62) between
distance traveled and all time active and inactive measures.
Pairwise correlations between all potential response variables
were non-zero (p <0.001). Due to its correlation with the
other response variables, total distance moved during the
five-minute observation period was selected as the single
representative behavior to be used in our statistical analyses
of odorant response.
Prior to statistical analysis, we reviewed data for visual and
statistical outliers in both pre-odor and post-odor distance
traveled. Given our data were generated using a tracking
software to analyze video recordings, we inspected the data to
ensure no outliers were included that may have resulted from
a tracking anomaly. Results from each trial were inspected
visually to identify abnormal and extreme values of distance
traveled by an individual. A Grubbs Test, which identified
extreme outliers from the assumed normal distribution of
the distance traveled measure in each treatment group, was
conducted using the outliers package (v0.14; Komsta, 2011)
to verify our visual observations. Two out of one hundred
and ninety-six trials were removed as outliers based upon
visual inspection and evaluation using the Grubbs Test. Four
additional trials were removed because of tracking related
issues (e.g. lighting variation) or incomplete video recordings.
We modeled post-odor distance traveled under a nor-
mal distribution using robust linear regression as a function
of predictor variables measured throughout the experiment.
Normality of residuals and homoscedasticity were assessed
following model selection for a traditional linear regression.
The model did not meet the assumptions. Specifically, we
observed multiple highly influential (high leverage) observa-
tions in our model based on the residual quantile-quantile and
residuals vs. leverage plots (Chatterjee and Hadi, 1986). Based
on these findings, we performed robust linear regression
models using an M estimator, which down-weighs highly
influential observations without removing observations from
the analysis (Filzmoser and Nordhausen, 2021). Models were
compared to select fixed effects to include in full model
predictions and inference. To account for individual varia-
tion in swimming behaviors and activity, pre-odor distance
traveled was included as a predictor variable in all but the
null model. The fixed effects included individual length, pre-
odor distance traveled, treatment group and their pairwise
interactive effects. Models were compared using Akaike Infor-
mation Criterion—small sample size correction (AICc) with
the AICcmodavg package (v2.3–1; Mazerolle, 2020). All
models within two AICc of the top model were considered
(Tredennick et al., 2021).
Robust linear mixed models were run based on results from
the fixed effects model selection using the robustlmm package
(Koller, 2016). Models also included arena as a random
intercept, tank as a random intercept, or both arena and tank
as crossed random effects to account for non-independence
of individuals based on these factors. However, the estimated
effect of tank was 0 so only the random effect of arena was
included to prevent overfitting. Random effects were included
for model interpretation but not selection, as we were not
interested in making inferences on the random effects (Gomes,
2022) and there is no accurate method for robust linear mixed
model comparisons (Koller, 2016). Figures were produced
for the focal model using the ggplot2 (Wickham, 2016) and
cowplot (v1.1.1; Wilke, 2020) packages. Predictions based on
robust linear models were made using the predict.rlm function
from the MASS package and included only fixed effects
because the predict.rlm function cannot generate confidence
intervals for mixed models (Venables and Ripley, 2002).
Results
Lake sturgeon reared in water containing PEA (1.04 x
10−7M) and morpholine (9.90 x 10−11 M) during early
life stages traveled a greater distance after exposure to
the odorants in behavioral experiments compared to naïve
(control) individuals. AICc values indicated the best-fit model
included pre-odor distance traveled, treatment and their
interaction (Table 1). Pre- and post-odor distance traveled
were positively correlated across all treatments, though
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Research article Conservation Physiology • Volume 11 2023
Tab le 1 : Comparison of AICc scores of robust linear regression models for post-odor distance traveled responses associated with dierent
xed-eects
Model AICc
Treatment +pre-odor distance +treatment∗pre-odor distance 0
Treatment +pre-odor distance +length +treatment ∗pre-odor distance 2.71
Pre-odor distance 3.53
Length +pre-odor distance +length ∗pre-odor distance 5.42
Length +pre-odor distance 5.48
Treatment +pre-odor distance 13.34
Treatment +pre-odor distance +length 15.57
Treatment +pre-odor distance +length +length ∗pre-odor distance 16.94
Treatment +pre-odor distance +length +treatment ∗pre-odor distance +length ∗pre-odor distance +treatment ∗length 18.26
Treatment +pre-odor distance +length +treatment ∗length 23.4
Intercept only 184.45
AICc scores are calculated in referenceto the model with the lowest AICc. Fixed eects included treatment, pre-odor distance, total length of the individual and pairwise
interactions between the independent variables.
this relationship varied by treatment (Fig. 2A;Table 2). All
groups exposed to PEA and morpholine, except for the group
exposed only during the juvenile stage, had a larger increase
in post-odor distance traveled with a unit increase in pre-odor
distance traveled (slope) compared with fish never exposed
to PEA or morpholine prior to behavioral testing. Predicted
distances traveled and 95% confidence intervals based
upon our best-fit model, and a constant pre-odor distance
(2635 ±163 cm; mean ±SE) indicated the exogenous feeding
and free-embryo stages were most important (Fig. 2B); fish
exposed during the exogenous feeding stage only had the
highest predicted post-odor distance traveled response at
the mean pre-odor distance traveled (56% larger than the
control), followed by fish exposed during the free-embryo
stage only (42% larger than control). Predicted post-odor
distance traveled responses at the mean pre-odor distance
and slope estimates were not larger for fish exposed during
consecutive stages when compared to fish exposed during the
free-embryo or exogenous feeding stages only (Fig. 2B).
Discussion
As discussed below, our study does did not directly inves-
tigate adult homing via olfactory imprinting during earlier
life stages, as we only studied behavioral responses of juve-
niles. Nevertheless, the results support the hypothesis that
lake sturgeon imprint to odors experienced during early life
stages. Age-0 lake sturgeon reared in water activated with
PEA and morpholine for as little as 7 days responded to the
odorant mixture in behavioral (movement distance) assays
over 50 days after the initial odorant exposure. The crit-
ical imprinting window included the free-embryo and the
exogenous feeding (larval) stages, as exposure to the odorants
during these periods resulted in elevated responses during sub-
sequent behavioral testing. In contrast, exposure during egg
or juvenile stages yielded responses no different than control
treatments in which fish were naïve to the odorants prior to
behavioral testing. Taken together, the results provide the first
direct evidence for olfactory imprinting in lake sturgeon, and
implicate the free-embryo and larval stages as developmental
periods important for imprinting.
Experimental evidence for embryonic and larval imprint-
ing aligns with established aspects of lake sturgeon ecology
and developmental biology (Zhang and Dang, 2014). Age-0
lake sturgeon remain close to stream spawning and hatching
locations through the onset of exogenous feeding (13–19 days
after hatch). Subsequently, downstream dispersal begins and
juveniles leave streams within ∼4 months (Holtgren and
Auer, 2004;Benson et al., 2005;Peterson et al., 2007). Unlike
species of Pacific salmon that remain in their natal stream
for over a year and can imprint after age 1 (Dittman et
al., 1996), lake sturgeon must imprint to stream odors dur-
ing early life stages if these odors guide natal homing by
sexual mature adults some 15–20+years later (Peterson et
al., 2007). Notably, previous morphological studies on age-
0 lake sturgeon indicate rapid development of the olfactory
bulb during the transition from free-embryos to exogenously
feeding larvae (Zhang and Dang, 2014). In Russian sturgeon,
physiological and behavior studies provide evidence for olfac-
tory imprinting and elevated thyroid hormones during larval
stages (Boiko and Grigor’yan, 2002;Boiko et al., 2004). Our
study provides behavioral evidence for olfactory imprinting
during the free-embryo and larval stages in lake sturgeon,
though it does not exclude the possibility of additional win-
dows of imprinting later during the juvenile stage because
we only attempted to imprint fish up to approximately two
months post-fertilization.
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Conservation Physiology • Volume 11 2023 Research article
Figure 2: Slope estimates for the relationship between pre-odor
distance traveled (A) and predicted post-odor distance traveled at
the mean of 2635 cm after 1000 simulations (B) based on the robust
linear model relating post-odor distance to treatment, pre-odor
distance and the interaction between treatment and pre-odor
distance, which was identied as the top model based upon AICc
values (see Table 1). Error bars represent one SE of the slope
estimates (A) and 95% condence intervals of predicted responses
(B). Red dashed horizontal lines were included for comparison
between the control and other treatment groups and represent the
estimated slope value and predicted post-odor distance for the
control. Black dashed horizontal lines represent the value of the
lower and upper 95% condence intervals of the post-odor distance
prediction for the control group. Numbers above the estimates
represent sample sizes for each treatment.
Whether the free-embryo and larval stages represent one
continuous or two distinct imprinting periods in lake sturgeon
remains unclear. Juvenile lake sturgeon responded to the
artificial odorants after being reared in them during the free-
embryo and larval stages combined or either stage individu-
ally. In Pacific and Atlantic salmon (Salmo salar), olfactory
imprinting occurs primarily during the parr-smolt transfor-
mation, which is an important period of behavioral, endocrine
and physiological changes (Hasler and Scholz, 1983;Morin
et al., 1989). Recent studies have also provided evidence for
olfactory imprinting at earlier stages (Dittman et al., 2015;
Bett et al., 2016;Armstrong et al., 2022), indicating olfactory
imprinting may occur sequentially at multiple developmental
stages and may guide natal homing not only to a specific river
but to a specific natal site (Quinn et al., 2006). Lake sturgeon
remain near the spawning site burrowed in the substrate at the
free-embryo stage (Kempinger, 1988) but leave the substrate
and drift downstream during the exogenous feeding larvae
stage (Auer and Baker, 2002), and could possibly imprint
to slightly different odors during each stage. Although fine-
scale natal homing has not been shown in sturgeon, observed
use of separate spawning sites by distinct groups of lake
sturgeon within a single river (Forsythe et al., 2012) could
be driven, in part, by imprinting during the free-embryo stage,
whereas imprinting during the larval stage could guide coarse-
scale homing to a river. Alternatively, the free-embryo and
larval stages could represent a single window during which
imprinting occurs, with exposure to an odor during only part
of the window sufficient for imprinting. Unlike Pacific salmon
(Havey et al., 2017), lake sturgeon did not show stronger
responses after exposure during two developmental stages
versus one. Additional research and an experimental design
that can directly compare responses to odors experienced
during each stage is needed to determine whether the free-
embryo and larval stages represent two distinct imprinting
windows.
Challenges associated with the life history of lake stur-
geon imposed constraints on our study that obfuscate the
ecological implications of our results. The 10- to 30-year pre-
reproductive stage of lake sturgeon (Peterson et al., 2007) pre-
cluded use of the approach often taken with Pacific salmon, in
which adults are tested for attraction to odorants they experi-
enced during early development (Bett et al., 2016). Observing
juvenile responses to imprinted odorants proved to be a useful
alternative that provided evidence for early life olfactory
imprinting, but the ecological function of juvenile responses
and the link to homing behavior of adults remain unknown.
Responses of age-0 fish to imprinted odors in other species are
related to kin recognition (zebrafish; Gerlach et al., 2008) and
larval settlement (coral reef fishes; Gerlach et al., 2007). In the
case of Pacific salmon, natal water preference by emergent fry
has been documented, though it is unclear what the ecological
function of this response is and how this behavior relates
to the natal homing behaviors in adult salmon (Bodznick,
1978;Dittman et al., 2015). Our behavioral assays were
not designed to evaluate the ecological function of juvenile
responses to imprinted odorants in lake sturgeon; however,
the observed increase in total distance traveled after exposure
to imprinted odorants could conceivably relate to dispersal
of juveniles away from their natal habitat. Interestingly, a
previous study found that lake sturgeon stocked in Black
River were more likely to be recaptured downstream of the
release site if they were reared in, and therefore potentially
imprinted upon, Black River water versus water from an off-
site hatchery (Crossman et al., 2011). Although the observed
differences in recapture rates were likely due, at least in
part, to higher mortality of fish reared offsite (Crossman et
al., 2011), the results could also be explained by a greater
propensity of Black River-imprinted fish to disperse from
natal habitat downstream to novel feeding habitats. Future
research on olfactory imprinting in lake sturgeon could con-
tinue to leverage juveniles as a proxy of homing adults while
also elucidating the ecological function of juvenile responses
to imprinted odorants.
The results of our study inform efforts to manage lake
sturgeon, especially guidance pertaining to use of stream-side
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Research article Conservation Physiology • Volume 11 2023
Tab le 2 : Parameter estimates and SEs for the robust linear regression
model of post-odor distance traveled based on treatment group (e.g.
Free-Embryo [FE] or Juvenile [juv.]), pre-odor distance traveled, and the
interaction between treatment and pre-odor distance traveled
Parameter Estimate SE
Fixed-only
Intercept 521.229 283.05
Egg −254.647 424.32
FE −720.232 427.00
Larvae −802.955 418.29
Juv. 423.184 402.24
Egg-FE −360.368 404.10
Egg-FE-larvae −311.486 394.62
Egg-FE-larvae-juv. −108.347 430.44
FE-larvae-juv. 480.305 393.42
Larvae-juv. 276.980 393.30
Pre-odor dist. 0.440 0.10
Egg ∗pre-odor dist. 0.223 0.16
FE ∗pre-odor dist. 0.543 0.14
Larvae ∗pre-odor dist. 0.662 0.14
Juv. ∗pre-odor dist. −0.045 0.12
Egg-FE ∗pre-odor dist. 0.366 0.13
Egg-FE-larvae ∗pre-odor dist. 0.372 0.13
Egg-FE-larvae-juv. ∗pre-odor dist. 0.180 0.14
FE-larvae-juv. ∗pre-odor dist. 0.118 0.12
Larvae-juv. ∗pre-odor dist. 0.106 0.12
Fixed +Arena as a random intercept
Intercept 858.464 329.28
Egg −417.863 435.75
FE −877.482 438.37
Larvae −1080.007 433.89
Juv. 281.279 418.70
Egg-FE −569.650 417.14
Egg-FE-larvae −498.138 405.07
Egg-FE-larvae-juv. −423.665 441.74
FE-larvae-juv. 312.664 403.80
Larvae-juv. 144.816 403.88
Pre-odor dist. 0.246 0.10
Egg ∗pre-odor dist. 0.339 0.16
FE ∗pre-odor dist. 0.683 0.15
Larvae ∗pre-odor dist. 0.807 0.14
(Continued)
Tab le 2 : Continued
Parameter Estimate SE
Juv. ∗pre-odor dist. 0.102 0.13
Egg-FE ∗pre-odor dist. 0.518 0.13
Egg-FE-larvae ∗pre-odor dist. 0.515 0.14
Egg-FE-larvae-juv. ∗pre-odor dist. 0.388 0.14
FE-larvae-juv. ∗pre-odor dist. 0.264 0.13
Larvae-juv. ∗pre-odor dist. 0.254 0.12
Estimates on the left are from the xed-eects only models and estimates on the
right are from a robust linear mixed model with the arena used for observations
as a random intercept.
rearing facilities (Holtgren et al., 2007). First, our experiments
provided behavioral evidence that lake sturgeon can imprint
to odors experienced during early life stages, a critical pre-
diction of the hypothesis that the genetic structure of lake
sturgeon results from natal homing via imprinted stream
odors (DeHaan et al., 2006;Welsh et al., 2008;Homola et al.,
2012;Donofrio et al., 2018;Scribner et al., 2022). Although
stream-side rearing facilities also increase survival of age-
0 lake sturgeon (Crossman et al., 2011), a major objective
of their use is to imprint lake sturgeon to targeted rivers
and encourage adult homing to those rivers (Holtgren et al.,
2007). Second, evidence reported here and elsewhere (Boiko
and Grigor’yan, 2002;Boiko et al., 2004;Zhang and Dang,
2014) suggesting that olfactory imprinting occurs during
the free-embryo and larval stages implies imprinting lake
sturgeon to targeted rivers may be most effective when fish
are brought into stream-side rearing facilities during early life
stages. Notably, many stream-side rearing facilities raise lake
sturgeon captured as drifting larvae in the target stream or
from other higher-producing streams (Holtgren et al., 2007).
Our results suggest these fish may have already begun to
imprint as free-embryos, which could affect the success of
olfactory imprinting to other streams. Additional research is
needed to determine the specific roles of imprinting during
the free-embryo versus larval stages, especially for stream-side
rearing facilities that rely on lake sturgeon caught as larvae
from other rivers.
In conclusion, we provide behavioral evidence for embry-
onic and larval imprinting to odorants in a non-teleost with
a life history markedly different from Pacific salmon. Impor-
tantly, we addressed only one prediction of the hypothesis that
olfactory imprinting guides natal homing in lake sturgeon—
the ability to respond to odorants experienced during specific
life stages. However, numerous aspects of the sturgeon life
cycle strain the model of natal homing via olfactory imprint-
ing that has been developed for Pacific salmon (Dittman et
al., 2015). In particular, the delayed age-at-maturity raises
questions about the stability of a stream’s signature odor and
the persistence of lake sturgeon’s memory for it. Likewise,
migratory patterns differ among populations of lake sturgeon
and some migratory behaviors, such as moving downstream
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Conservation Physiology • Volume 11 2023 Research article
into lake outflows to spawn (Kessel et al., 2018), are unlikely
guided by olfactory cues in the same way as the migratory
behaviors of Pacific salmon. Continued research on lake
sturgeon is likely to inform conservation of one of the world’s
most imperiled families of fish (Billard and Lecointre, 2000),
and contribute to a model of olfactory imprinting that is more
generalizable across diverse fish species.
Funding
This work was supported by the Great Lakes Fishery Trust
[project ID 1785].
Author Contributions
J.G.K. collected and analyzed data, prepared figures and
led manuscript writing. D.L.L assisted in data collection.
T.J.B., E.A.B., T.G.Z., K.T.S. and W.L. procured funding. All
authors helped with conceptualization of the study, method-
ology development and assisted in writing and editing of the
manuscript.
Data availability
The data underlying this article are available at the Dryad
Digital Repository https://doi.org/10.5061/dryad.0p2ngf25t.
Acknowledgements
We acknowledge Maxwell Majinska, Jessie Hanson, Emily
Mayer, BayLee Moser, Alex Florian, Kayla Reed and Rachael
Kermath for their efforts in maintaining the fish and conduct-
ing behavior experiments.
Supplementary Material
Supplementary material is available at Conservation Physiol-
ogy online.
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