Draft...In frogs, the development of the olfactory system throughout metamorphosis has been well...

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Olfactory epithelium ontogenesis and function in postembryonic North American bullfrog tadpoles

(Lithobates catesbeiana)

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2019-0213.R1

Manuscript Type: Article

Date Submitted by the Author: 21-Nov-2019

Complete List of Authors: Heerema, Jody; University of Lethbridge, Department of Biological SciencesBogart, Sarah; University of Lethbridge Department of Biological Sciences, Helbing, Caren; University of Victoria, Department of Biochemistry & MicrobiologyPyle, Greg; University of Lethbridge, Department of Biological Sciences

Is your manuscript invited for consideration in a Special

Issue?:Not applicable (regular submission)

Keyword:Lithobates catesbeiana, North American bullfrog tadpole, OLFACTION < Organ System, BEHAVIOUR < Discipline, METAMORPHOSIS < Discipline, EOG

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

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Title: Olfactory epithelium ontogenesis and function in postembryonic North American

bullfrog tadpoles (Lithobates catesbeiana)

Authors: J. L. Heerema1, S. J. Bogart1*, C. C. Helbing2, G. G. Pyle1

Author affiliations:

1Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada

2Department of Biochemistry and Microbiology, University of Victoria, BC, Canada

Author emails: JLH: [email protected]; SJB: [email protected];

CCH: [email protected]; GGP: [email protected]

Author ORCID iDs: JLH: 0000-0002-2202-8819; SJB: 0000-0002-2313-8649;

CCH: 0000-0002-8861-1070; GGP: 0000-0003-4338-7674

*Corresponding author: Sarah J. Bogart, Dept. of Biological Sciences, University of Lethbridge,

4401 University Drive, Lethbridge, AB, Canada, T1K 3M4. Phone: 403-332-4048. E-mail:

[email protected]

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mailto:[email protected]





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Title: Olfactory epithelium ontogenesis and function in postembryonic North American

bullfrog tadpoles (Lithobates catesbeiana)

Authors: J. L. Heerema1, S. J. Bogart1*, C. C. Helbing2, G. G. Pyle1

Abstract

During metamorphosis, the olfactory system remodelling in anuran tadpoles—to transition from

detecting waterborne odorants to volatile odorants as frogs—is extensive. How the olfactory

system transitions from the larval to frog form is poorly understood, particularly in species that

become (semi-)terrestrial. We investigated the ontogeny and function of the olfactory epithelium

of Lithobates (Rana) catesbeiana Shaw 1802 tadpoles at various stages of postembryonic

development. Changes in sensory components observable at the epithelial surface were examined

by scanning electron microscopy (SEM). Functionality of the developing epithelium was tested

using a neurophysiological technique (electro-olfactography; EOG), and behaviourally, using a

choice maze to assess tadpole response to olfactory stimuli (algae extract, amino acids). The

youngest (premetamorphic) tadpoles responded behaviourally to an amino acid mixture despite

having underdeveloped olfactory structures (cilia, olfactory knobs) and no EOG response. The

consistent appearance of olfactory structures in older (prometamorphic) tadpoles coincided with

reliably obtaining EOG responses to olfactory stimuli. However, as tadpoles aged further, and

despite indistinguishable differences in sensory components, behavioural- and EOG-based

olfactory responses were drastically reduced, most strongly near metamorphic climax. This work

demonstrates a more complex relationship between structure and function of the olfactory system

during tadpole life history than originally thought.

Key words: Lithobates catesbeiana, North American bullfrog tadpole, olfaction, behaviour,

metamorphosis, EOG

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Introduction

The postembryonic development of anurans includes the metamorphosis of the free-

living aquatic, herbivorous tadpole to a (semi-)terrestrial, carnivorous frog (Gilbert et al. 1996).

During metamorphosis, there are major developmental changes in the anuran olfactory system as

it shifts from functioning exclusively in water environments in tadpole larvae to functioning in

both water and air environments as frogs (reviewed in Gascuel and Amano 2013). These

metamorphic changes may affect olfactory detection and response to food and predators by

larval anurans.

In the broadest sense, olfactory detection occurs at the surface of the olfactory

epithelium. The olfactory epithelium is composed of three cell types, which we will describe

briefly: sustentacular cells (SCs), which provide physical and metabolic support for the

epithelium, phagocytose dead neurons, and can help modulate odorant detection (reviewed in

Getchell and Getchell 1992, and Schwob 2002; Lucero 2013); basal (progenitor) cells, which are

involved in the maintenance and regeneration of the epithelium; and olfactory sensory neurons

(OSNs), which function to detect odours (reviewed in Manzini and Schild 2010). The OSNs are

bipolar neurons that project either cilia or microvilli—both richly supplied with olfactory

receptors (ORs)—to the apical surface of the epithelium (reviewed in Schwob 2002, and Manzini

and Schild 2010). Logically, if the surface structures of OSNs are underdeveloped, the olfactory

sense may be impaired.

In frogs, the development of the olfactory system throughout metamorphosis has been

well studied in Xenopus laevis Daudin 1802 (African clawed frog), but other species remain less

well documented (Dittrich et al. 2016; Syed et al. 2017). Although there are interspecies

differences in the olfactory system on a cellular level, the overall structural development is

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conserved (Gascuel and Amano 2013). Generally, the larval olfactory epithelium originates

from a placode, which gives rise to an olfactory pit (Klein and Graziadei 1983). Each olfactory

pit differentiates into a principal cavity (PC) and vomeronasal organ (VNO) (Hansen et al. 1998).

In the tadpole, the olfactory epithelium lies within the PC and contains both ciliated OSNs and

microvillous OSNs, which are specialized to detect only waterborne odorants (Hansen et al.

1998). The VNO contains microvillous OSNs and is presumed to be involved in the detection of

pheromones (conspecific signalling odorants; reviewed in Døving and Trotier 1998), but may

have other functions. As development proceeds towards metamorphosis, the middle cavity (MC)

is formed de novo between the PC and VNO (Hansen et al. 1998; Dittrich et al. 2016) and the PC

olfactory epithelium is remodelled extensively (Hansen et al. 1998). After metamorphosis, the

PC contains only ciliated OSNs (Mezler et al. 1999) and is specialized to detect volatile odours,

which are typical of a terrestrial environment (Higgs and Burd 2001; Gascuel and Amano 2013).

In most species, the MC contains only non-sensory epithelium. However, in the case of X.

laevis, the adult MC resembles the larval PC, and contains both microvillous OSNs and ciliated

OSNs, and functions to detect odours in aquatic environments (Hansen et al. 1998; Wang et al.

2008; Gascuel and Amano 2013; Dittrich et al. 2016). The frog VNO does not change from the

tadpole form and continues to contain only microvillous OSNs (Taniguchi et al. 1996; Hansen et

al. 1998). It should be noted that in contrast to the majority of anurans, X. laevis remains aquatic

in adult form (despite it also becoming air-breathing). Thus, due to interspecies differences, X.

laevis may not be a true model of olfactory development for other anurans undergoing

metamorphosis, especially regarding their olfactory-mediated function and behaviours.

The development of the olfactory system and many other tissues during metamorphosis is

driven by thyroid hormones (THs) thyroxine (T4) and 3,3’,5-triiodothyronine (T3) (Brown and

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Cai 2007). Metamorphic changes include cell apoptosis resulting in tail resorption and loss of

gills; organogenesis of limbs, stomach, and bone marrow; and the complete remodelling of the

intestine, brain, and spinal cord (Tata 2006; Brown and Cai 2007). Metamorphosis is divided

into three stages with respect to endogenous TH concentrations. During premetamorphosis, the

thyroid gland is inactive and no TH circulates through the anuran body. During

prometamorphosis, the thyroid gland becomes active and begins secreting TH and initiating

metamorphic changes. At metamorphic climax, endogenous TH is at its highest concentration,

triggering extensive changes and remodelling throughout the anuran body (reviewed in Tata,

2006; Brown and Cai 2007). Given the substantial changes that occur in the tadpole olfactory

system during development, changes in chemosensory function, including olfaction, are

expected. However, very little is known about the nature of these structural and functional

changes in frogs that more completely transition to a terrestrial existence, such as in the North

American bullfrog, Lithobates (Rana) catesbeiana Shaw 1802.

At a functional level, previous studies have utilized chemosensory cues to evoke tadpole

behavioural responses in a variety of species using many different assays (Mirza et al. 2006;

Smith et al. 2008; Takahara et al. 2012). Two common approaches to studying chemosensation,

particularly olfaction, are to either use calcium imaging (Sachse and Galizia 2002) or to measure

the odour-evoked extracellular field potentials using electro-olfactography (EOG) (reviewed by

Scott and Scott-Johnson 2002). Early studies on Lithobates pipiens Schreber 1782 frogs

demonstrated the utility of EOG measurements for defining electrical responses to a series of

stimuli (Getchell 1974). Odour molecules interact with ORs on the cilia or microvilli of OSNs,

triggering a molecular signal transduction cascade that leads to bulk depolarization of the OSNs.

The EOG measures the loss of cations from the extracellular environment (i.e. at the surface of

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the olfactory epithelium) resulting from this bulk depolarization. The amplitude of the resulting

electro-olfactogram is related to the magnitude of the bulk depolarization of the OSNs after

odorant stimulation, thus indicating the magnitude of the olfactory response. Previous studies

have used EOG to measure the olfactory response of fish to odorants (Green et al. 2010; Dew et

al. 2014), and the technique has recently been modified for use with L. catesbeiana tadpoles

(Heerema et al. 2018a).

The purpose of the present study was to investigate ontogenetic changes in the L.

catesbeiana tadpole olfactory system during metamorphosis, as measured by changes in the

surface ultrastructure of the olfactory epithelium and in the olfactory-based, neurophysiological

and behavioural responses to odorants. Overall, we hypothesize that 1) changes in olfactory

structures during tadpole development will lead to measurable differences in neurophysiological

responses and ultimately alter tadpole behaviour. We used scanning electron microscopy (SEM)

to compare the surface ultrastructure of the olfactory epithelium, EOG to examine for

measurable olfactory responses at the neurophysiological level, and behavioural choice maze

tests to elucidate any behavioural changes in tadpoles, at various stages of post-embryonic

development. For this study, tadpoles were grouped into previously published, developmental

groups that are classified according to differences in the concentration of endogenous TH and

outwardly recognizable markers of development (i.e. premetamorphosis, prometamorphosis, and

nearing metamorphic climax are marked by limb development). Additional investigations were

conducted within these developmental groups, as needed, to further understand variations in

structure, function, and behaviour.

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Materials and Methods

Animals

Lithobates catesbeiana (Shaw, 1802) tadpoles of mixed sex were wild caught in Victoria,

BC, Canada, by Westwind Sealab Supplies under permit from the Capital Regional District

Parks and Recreation. Tadpoles were housed for two weeks in dechlorinated city water in a re-

circulatory system in the Aquatic Research Facility (ARF) at the University of Lethbridge, AB,

Canada, prior to experimentation. Housing conditions included 15°C water temperature on a

light: dark 16:8 h photoperiod and tadpoles were fed ad libitum daily with Spirulina spp. Turpin

Ex Gomont, 1893 algae flakes. Prior to experiments tadpoles were fasted and temperature-

acclimated to 24 °C for 72 hours in aerated, dechlorinated water in 15 L polypropylene buckets.

During the temperature acclimation water quality including temperature (mean, range; 24.2, 21.4

– 25.0 °C, n = 16), dissolved oxygen (mean, range; 78.9, 46.7 – 94.7 %; 6.7, 4.1 – 7.9 mg/L, n =

15) was measured daily and pH (median, range; 8.27, 7.79 – 8.39, n = 9) every other day.

Hardness (mean ± SE; 178 ± 1 mg/L as CaCO3) and alkalinity (mean ± SE; 136 ± 2 mg/L as

CaCO3, n = 30) are routinely measured in the ARF and are stable through time. Tadpoles were

staged according to Taylor and Kollros (1946) and will be referred to by developmental group

for ease of comparison according to Table 1. All procedures involving tadpoles were approved

by the Animal Welfare Committee at the University of Lethbridge (protocol #1401) and the

University of Victoria Animal Care Committee (protocol #2011-030), for compliance under the

Canadian Council on Animal Care guidelines.

<Table 1 placement>

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Experimental design

The initial development of the olfactory sense was examined using the youngest tadpoles.

Specifically, the EOG response of premetamorphic and early prometamorphic tadpoles (mean ±

SE body mass: 7.0 ± 0.4 g; median, range: TK VI, I – XIII, n = 7) was measured to examine the

ontogenetic progression of a measurable olfactory response to a food cue (Spirulina extract;

algae). To determine whether olfaction was used to behaviourally respond to chemical stimuli at

early stages, premetamorphic tadpoles (mean ± SE body mass 4.6 ± 0.28 g; median, range: TK

IV, I – VIII, n = 10 - 12) were rendered anosmic and their response to an olfactory stimulus was

tested in a choice maze relative to unaffected animals. In this test, an amino acid mixture was

used as the stimulus because premetamorphic tadpoles did not respond to Spirulina in

preliminary behavioural tests (see section on preparation of olfactory stimuli). To link the ability

to obtain EOG responses to the ontogenetic appearance of key sensory structures (cilia and

olfactory knobs) on the surface of the olfactory epithelium, olfactory pits from premetamorphic

tadpoles used in EOG measurements were excised and examined by SEM.

Later stage tadpoles were primarily examined for ontogenetic changes in olfactory

function and behaviour as metamorphosis progressed. Behavioural attraction responses to

Spirulina extract were measured in early prometamorphic tadpoles (mean ± SE mass 10.1 ± 0.9

g; median, range TK: XII, XI –XII, n = 9) using a linear trough-style choice-maze. Later

developmental stage tadpoles (mean ± SE mass 22.5 ± 1.1 g, n = 23) were divided into two

experimental groups: late prometamorphic and tadpoles approaching metamorphic climax (n =

10 – 11). For these groups, behavioural responses to Spirulina extract were recorded in the

linear trough-style choice maze. Then, EOG responses to Spirulina extract and an amino acid (L-

alanine) were measured on the same tadpoles. Lastly, olfactory pits were excised from these

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individuals and observed by SEM. Details of all preparations and procedures are as described in

the sections below.

Preparation of olfactory stimuli

All olfactory stimuli used in this study were previously established as appropriate cues to

study tadpole behaviour and neurophysiological responses (Manzini et al. 2002; Hassenklöver et

al. 2012; Heerema et al. 2018a, 2018b). The type and concentration of cues used in EOG and

behaviour experiments was dependent upon the ability to elicit consistent responses to the

stimuli, at the lowest concentration possible, as previously described by Heerema et al. (2018a,

2018b). Preliminary testing of lower concentrations of stimuli described below did not elicit

consistent behavioural responses in the tadpoles. Stimuli included Spirulina (algae) extract and

an amino acid (L-alanine), representing food cues, or an equimolar amino acid mixture (L-

alanine, L-serine, glycine), representing a predator cue. To prepare the Spirulina extract for

behavioural and EOG experiments, Spirulina flakes (2 g/L; Nutrafin Max, Hagen, Montreal,

Canada) were added to dechlorinated water, stirred for 30 minutes, and then filtered through

aquarium filter floss (Aqua-Fit, Hagen, Montreal, Canada). For EOG experiments, 10-2 M L-

alanine (USP grade, Sigma-Aldrich, Oakville, Canada) was dissolved in dechlorinated water.

Due to a lack of consistent behavioural responses to Spirulina and to single amino acids in

preliminary testing of premetamorphic tadpoles, an amino acid mixture was used instead

(Heerema et al. 2018a, 2018b). To prepare the amino acid mixture, equimolar concentrations

(0.022 M each) of L-alanine (USP grade, Sigma-Aldrich, Oakville, ON, Canada), L-serine (USP

grade, VWR, Radnor, USA), and glycine (proteomics grade, AMRESCO, Cleveland, USA) were

dissolved into dechlorinated water, producing a mixture concentration of 0.066 M. It should be

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noted that the stimuli concentrations reported here for use in behavioural tests are stocks and do

not reflect the final concentrations in the testing maze (see the section Behavioural choice maze

assays for details). All stimuli were prepared fresh daily.

Electro-olfactography (EOG)

Methodology of Heerema et al. (2018a) was used to conduct EOG on L. catesbeiana

tadpoles wherein tadpole olfactory responses to olfactory stimuli (cues; 2 g/L Spirulina extract

and 10-2 M L-alanine) were measured. In short, tadpoles were anaesthetized in buffered tricaine

methanesulfonate (500 mg/L; TMS, Aqua Life, Syndel Laboratories, Nanaimo, Canada) until the

heartbeat slowed to approximately one beat every two seconds. Preparation and placement of

the EOG electrodes were as previously described by Heerema et al. (2018a). Olfactory stimuli,

without dilution, were individually delivered to the exposed olfactory pit in pulses of five

seconds and a minimum of two minutes were allowed to pass between each stimulus delivery to

prevent habituation. The delivery order of the cues was randomized and the response to each cue

was measured three times throughout the EOG test. Responses to each stimulus were averaged

for each individual. Mean EOG responses to olfactory stimuli were blank corrected by

subtracting any response elicited by the blank across each individual.

Inducing anosmia

To induce anosmia, tadpoles were anesthetised in buffered TMS (500 mg/L) and the

olfactory pits were exposed in the same manner used for tadpole EOG (Heerema et al. 2018a).

Surgical glue (Vetbond, 3M Canada, London, Canada) was applied to the olfactory pits,

completely capping them, and allowed to dry for approximately 30 seconds, hence rendering the

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tadpole anosmic by blocking external access to the neurons but without removing them (i.e. the

least invasive option). The tadpoles were placed in dechlorinated water and allowed to recover

for 48 h before their behavioural responses to an olfactory stimulus was challenged in a choice

maze. The lack of response to an olfactory stimulus confirmed anosmic condition (see results

section).

Behavioural choice maze assays

To measure tadpole behavioural responses, the choice maze test protocol of Dew et al.

(2014) was used with a few species-specific modifications made for animal size. Linear troughs

(75 cm x 20 cm x 15 cm; l x w x h), representing the choice maze, were divided lengthwise into

three zones (see Dew et al., 2014 for maze illustration). The two distal zones served as areas to

receive olfactory stimuli (stimulus-delivery zones) while the middle section served as the

acclimation zone. Placed within the acclimation zone was a clear plastic, bottomless container

(21.5 cm x 15 cm x 11.5 cm; l x w x h), serving as an acclimation chamber. This chamber

allowed tadpoles to adjust to maze conditions while it prevented their access to either of the

stimulus-delivery zones during the acclimation period, which immediately preceded each

behavioural trial.

For each trial, 8 L of dechlorinated water was added to the maze. A single tadpole was

placed in the acclimation chamber and allowed to acclimate to maze conditions for 20 min prior

to the start of the choice assay. Then, to each stimulus-delivery zone, 50 mL of blank

(dechlorinated water) or stimulus (2 g/L of Spirulina extract or an equimolar mixture of L-

alanine, L-serine, and glycine; total molarity 0.066 M) were remotely administered via silicone

tubing and syringes. Once administered to the maze, the cue and blank were allowed to diffuse

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for one minute (as determined in odorant diffusion tests) before the acclimation chamber was

remotely lifted via an overhead pulley system. The lifted acclimation chambers allowed the

tadpole to swim freely throughout the maze. Four tadpoles in separate mazes were tested

simultaneously, and the position of each tadpole in the maze was recorded every 10 sec for five

minutes. Time spent in each arm (stimulus zone) was calculated as in Dew et al. (2014). Note

that, the stimulus concentrations after complete cue dispersal in the stimulus delivery zone

(~3.14 L based on maze dimensions) were less than the administered concentration. The final

(dispersed) stimulus concentration in the maze would be approximately 0.001 M for the amino

acid mixture and approximately 0.03 g/L for Spirulina extract. The Spirulina extract did not

change the colour of the behavioural testing water, meaning that the tadpole visual system could

not be responsible for any resultant behaviour.

Several quality control measures were used in behavioural experiments. Without

animals, odorant diffusion tests were used to determine the diffusion time required for the cue

(50 mL of dechlorinated water mixed with food colouring) to reach the acclimation zone post

delivery. To ensure tadpoles did not inherently favour one arm of the maze, 50 mL of

dechlorinated water was administered to each arm of the maze and behavioural trials were run as

described above, prior to running behaviour experiments. The assignment of the blank or

olfactory stimulus to one end of the maze or other was randomized for each successive trial and

the researcher was blinded as to which end each stimulus was administered. To not influence

trials, all trials were recorded with a webcam (HD 720p, Logitech, Romanel-sur-Morges,

Switzerland) and observed on a laptop computer (MacBook Air, Apple, Cupertino, USA).

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Scanning electron microscopy (SEM)

Immediately following EOG experiments, tadpoles were euthanized in pH-buffered

tricaine methane sulfonate (MS-222; 1000 mg/L) solution. Olfactory pits were excised under a

dissecting microscope and fixed by immersion in Karnovsky’s fixative (Karnovsky 1965) for 24

h at room temperature. Fixed tissues were successively rinsed twice in cacodylate buffer (0.2 M;

pH 7.2; Ted Pella Inc., Redding, USA) for 15 min each, and dehydrated in a series of graded

ethanol dilutions (75% to 100% anhydrous; Commercial Alcohols, Brampton, Canada). Tissues

were dried and mounted on 15 mm aluminum stubs, sputter coated with platinum, and observed

with an SEM (S-3400N SEM, Hitachi, Tokyo, Japan) at the Lethbridge Research and

Development Centre, Lethbridge, AB, Canada. Entire olfactory pits were examined and imaged

via SEM in the youngest tadpoles. However, only the PC olfactory epithelium was imaged in

older tadpoles, which was differentiated from the VNO based on sensory cell types present (re

Døving and Trotier 1998; Hansen et al. 1998). SEM images were adjusted for brightness and

contrast using Adobe Photoshop CC v2015.5.0 and assembled into an image plate in Adobe

Illustrator CC v2015.3.0 (both Adobe Systems Inc., San Jose, USA).

Statistical Analyses

All statistical analyses were completed using R version 3.2.2 (R Core Team 2015), and R

Studio version 0.99.484 (R Studio Team 2015). For behavioural trials tadpoles that failed to

leave the acclimation chamber for the duration of the test were removed from the dataset, an a

priori decision. The average time spent in the stimulus arm and the control arm was compared

for each experimental group. Parametric assumptions were tested using the Shapiro-Wilk

normality test and Bartlett’s test of homoscedasticity on the paired differences of time spent in

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the stimulus and blank arms of the maze. Data meeting parametric assumptions were analyzed

with a paired t-test. Data not meeting parametric assumptions even after transformation were

analyzed with a paired Wilcoxon Rank Sum test. Power analyses and sample sizes were

calculated using the “effsize” package (Torchiano 2016). For behavioural experiments, the test

power of the behavioural test of the response of early prometamorphic tadpoles to odorants was

calculated as 0.74.

For EOG experiments, parametric assumptions were tested using the Shapiro-Wilk’s

normality test and the Bartlett’s test for homogeneity of variances. When parametric

assumptions were not met, data were transformed with a log10 (1 + x) transformation to reclaim

assumptions. Therefore, the transformed corrected mean EOG responses were compared with an

independent-samples t-test. Where parametric assumptions could not be reclaimed after

transformation, the Wilcoxon Rank Sum test was used to compare mean EOG responses. Mean

differences were considered to be significant when p ≤ 0.05.

Data availability

Data are available as part of the Supplementary Materials submitted with the manuscript.1

Results

Early stage development of olfactory structures vs EOG response

The surface morphology of the PC olfactory epithelium in premetamorphic tadpoles

changed considerably as metamorphosis progressed, and the appearance of sensory structures

(cilia and olfactory knobs) generally coincided with the ability to obtain EOG responses to

Spirulina extract and L-alanine (Fig. 1). At TK V (n = 7), the olfactory epithelial cells had a

1 Supplementary Materials, Tables S1, S2, S3, and S4.

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polygonal shape and short microvillous protrusions were evident (Fig. 1A, B), however, EOG

responses were not detectable at this stage (Fig. 1C). By TK VI, the polygonal cells were domed

and continued to bear short microvilli (Fig. 1D). A few specimens at TK VI were further

developed, with cilia from non-sensory cells (SCs) and mucus covering the epithelium (Fig. 1E).

These more developed TK VI specimens also exhibited the appearance of common sensory

structures, olfactory knobs with ciliated projections, which were commonly observed in later

stages (insets in Fig. 1G). Note that olfactory knobs, which are ~2 µm in diameter (Fig. 1G

insets), should not be confused with mucus appearing as larger globules, which obscured

underlying structures (Fig. 1E inset). Overall, EOG responses were undetectable at TK VI (Fig.

1F), although one individual did exhibit variable EOG responses to Spirulina extract. At TK IX,

olfactory knobs with cilia (ciliated OSNs) were consistently observed on the olfactory epithelium

(Fig. 1G). At TK IX, EOG responses to Spirulina extract and L-alanine were detectable (Fig. 1I)

but were still inconsistent across individuals. By early prometamorphosis (TK XIII), the

olfactory epithelium was densely covered by long cilia and mucus (Fig. 1H), and consistent

responses to Spirulina extract and L-alanine were measurable by EOG (Fig. 1I). Overall, ciliated

projections became longer and more prominent with increasing developmental stage, until they

completely covered the entire epithelial surface.

<Fig. 1 placement>

Anosmic vs control behavioural response of premetamorphic tadpoles

Tadpole response to the 0.001 M amino acid mixture (equimolar glycine, L-alanine, and

L-serine) was measured using tadpoles with intact olfactory pits and tadpoles that were rendered

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anosmic. When olfactory pits were left intact, tadpoles avoided the amino acid mixture,

spending significantly more time in the blank arm than the stimulus arm of the linear trough-

style choice maze (V = 51.5; p = 0.017; Fig. 2A). Even when the data were constrained to

tadpoles of age less than TK VI, i.e. before the first OSNs appeared (Fig. 1), the tadpoles

(median TK IV, range TK I - V; mean ± SE of 4.44 ± 0.40 g) still significantly avoided the cue

relative to the blank (V = 33.5, p = 0.035; Fig. S12). Conversely, there was no significant

difference between time spent in the blank and stimulus arms when premetamorphic tadpoles

were rendered anosmic (t11 = 0.81, p = 0.43; Fig. 2B).

<Fig. 2 placement>

Olfactory ontogeny in prometamorphic and older tadpoles

Attraction responses to Spirulina extract from early prometamorphic tadpoles were

measured with a linear trough-style choice maze (Fig. 3). Early prometamorphic tadpoles spent

significantly more time in the stimulus arm than the blank arm of the maze (t8 = -2.9, p = 0.01;

Fig. 3A).

<Fig. 3 placement>

Attraction responses from late prometamorphic (TK XVII) and tadpoles approaching

metamorphic climax (TK XVIII-XX) were also measured with the linear trough-style choice

maze (Fig. 3). Then, the same individuals were tested with EOG (Fig. 4) and their olfactory

epithelium surface anatomy was observed via SEM (Fig. 5). TK XVII tadpoles spent 3-fold

2 See Supplementary Materials

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more time in the stimulus arm than in the blank arm of the linear trough-style choice maze,

however this difference was not statistically significant (W = 6, p = 0.19; Fig. 3B). Tadpoles

approaching metamorphic climax (TK XVIII-XX tadpoles) were also not attracted to Spirulina

and spent the same amount of time in the stimulus and blank arms of the maze (t8 = -0.37, p =

0.71; Fig. 3C). Conversely, late prometamorphic tadpoles exhibited significantly higher EOG

responses to Spirulina extract than metamorphic climax tadpoles. Spirulina extract EOG

responses were over two-fold higher from late prometamorphic tadpoles when compared to

tadpoles approaching metamorphic climax (t21 = -2.4, p = 0.02, Power = 0.45; Fig. 4A). Late

prometamorphic tadpole EOG responses to L-alanine were also two-fold higher when compared

to tadpoles approaching metamorphic climax; however, this difference was not significant (W =

43, p = 0.16; Fig. 4B). SEM observation of the olfactory epithelium showed no qualitative

change in the sensory structures between late prometamorphic and tadpoles approaching

metamorphic climax. In all specimens observed, and similar to tadpoles at the TK XIII stage

(Fig. 1H), long ciliated projections and abundant mucus densely covered the olfactory epithelium

(Fig. 5).

<Fig. 4 placement>

<Fig. 5 placement>

Discussion

An ontogenetic shift in the olfactory system of L. catesbeiana tadpoles is evident on a

cellular and functional level throughout larval development. The results of the present study

demonstrate that discrete ontogenetic changes occur at specific stages of the larval period, as

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observed with EOG, behaviour, and scanning electron micrographs. To our knowledge, this is

the first study to compare olfactory responses between developmental stages in L. catesbeiana

with behaviour and EOG. Additionally, the surface anatomy of L. catesbeiana olfactory

epithelium has not previously been studied with SEM.

Early stage structural and functional development of the olfactory epithelium

The types of sensory structures observed here in bullfrog tadpoles are similar to those

observed in previous studies, which have mainly focused on X. laevis (Klein and Graziadei 1983;

Hansen et al. 1998). Polygonal-shaped cells observed in TK V – VI (Fig. 1A, B, D) in the

present study are similar to those Hansen et al. (1998) observed in the early developing PC of X.

laevis. We observed cilia and microvilli in tadpoles at all TK stages (Figs 1, 5), which were also

previously described by Hansen et al. (1998). In general, the cilia and microvilli observed in our

study could be projections from OSNs or SCs, since both of these cell types have been reported

in the larval PC in other species including X. laevis, Rana japonica Boulenger 1879, and

Ascaphus truei Stejneger 1899 (Taniguchi et al. 1996; Hansen et al. 1998; Benzekri and Reiss

2012). Although we did specifically identify OSNs (Fig. 1) that are required for an olfactory

response, additional histological investigation is warranted due to the presence of mucus that

obscured epithelial surface structures.

Previous studies on X. laevis have shown that not all cell types of the mature olfactory

epithelium are present at early developmental stages, but rather cells differentiate gradually

throughout development. For example, Klein and Graziadei (1983) and Hansen et al. (1998)

observed that microvilli develop before cilia in the PC, but both are present by Nieuwkoop and

Faber (NF) 39 (Nieuwkoop and Faber 1956), which precedes TK I. A similar progression of

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tissue development was also observed in R. japonica tadpoles (Taniguchi et al. 1996). Here, the

lack of detectable EOG olfactory responses to olfactory stimuli in early stage premetamorphic

tadpoles was seemingly due to the underdeveloped sensory structures in the olfactory epithelium.

The results (Fig. 1) clearly show that there is a close relationship between EOG responses to

olfactory stimuli and the cellular structures present on the olfactory epithelium, including cilia

and olfactory knobs. Although a few specimens at TK VI and all specimens at TK IX had

developed olfactory knobs and sparse cilia, EOG responses were variable (Fig. 1D, E, F, G, I).

Comparatively, at TK XIII, the dense cover of cilia over the olfactory epithelium resulted in

consistent EOG responses (Fig. 1H, I). However, the lack of EOG responses to olfactory stimuli

at developmental stages earlier than TK VI in our study brings into question when exactly the

olfactory epithelium begins to function.

For X. laevis, Hansen et al. (1998) suggests the olfactory system begins functioning at

approximately NF 45 (which precedes TK I), when OSNs first become functional and the

tadpole begins feeding. Given the various staging regimes used, it can be difficult to compare

both the developmental stage (McDiarmid and Altig 1999) and the characteristics that might be

unique to specific stages across species. Even considering these discrepancies, the tadpoles used

in the present study were more developed than those in Hansen et al. (1998). Therefore, the

olfactory epithelium of tadpoles in the present study should have been functioning.

However, the overall lack of EOG responses at early stages (≤ TK VI) in L. catesbeiana

tadpoles in the present study suggested that the olfactory epithelium was not yet functional.

Nevertheless, when tested in a linear trough-style choice maze, TK I – VIII tadpoles with their

olfactory senses intact avoided the olfactory stimulus (Fig. 2A and Fig. S13), but those rendered

anosmic (by occluding their entire nares with glue) did not exhibit the same avoidance response

3 Ibid

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to the olfactory stimulus (Fig. 2B and Fig. S14). Therefore, intact premetamorphic L.

catesbeiana tadpoles of age less than TK VI must have had an alternate, functional

chemosensory system in their nasal cavity and were using it to respond to cues since there was

no visual evidence of developed OSNs until tadpoles were at least of stage TK VI (Fig. 1). One

possibility is that there are solitary chemosensory cells (SCCs) present in the epithelial lining of

the nasal cavity of premetamorphic L. catesbeiana tadpoles. SCCs are sensory cells embedded

in epithelial tissues of vertebrates, including in the skin of tadpoles of other ranid species (e.g.

Rana temporaria L.), and in the nasal epithelium of American alligators (Alligator

mississippiensis Daudin, 1802), that are not associated with the olfactory nerve but are instead

linked to fibers of the trigeminal nerve (Whitear 1976; Kotrschal 1991; Hansen 2007; and

references therein). Like OSNs, SCCs can respond to chemosensory cues. When present, SCCs

sparsely populate epithelial tissue relative to other cells and are difficult to positively identify

using light microscopy or SEM without validation by techniques such as transmission electron

microscopy (TEM) and immunocytochemistry (Hansen 2007).

Moreover, the aforementioned olfactory-driven behavioural response in premetamorphic

tadpoles also indicates that early stage olfactory sensory structures are not developed enough to

generate a detectable EOG response and second, EOG analysis is not sensitive enough to record

olfactory responses at early developmental stages. It is plausible that a low density of olfactory

sensory structures at these early developmental stages, as observed in X. laevis larvae (Hansen et

al. 1998), resulted in the absence of EOG response in our study. Finally, the broad range of

anatomical development and EOG responses observed in TK VI individuals is likely due to

natural biological variation. This variation may be a sign of precocious metamorphic

development and suggests that there is limited fidelity between external markers of tadpole age

4 Ibid

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(i.e. small changes in limb development; Taylor and Kollros 1946) and timing of olfactory

remodelling, particularly around TK VI.

Shift in olfactory function in older tadpoles

Further ontogenetic changes in olfactory function were observed in L. catesbeiana

tadpoles in later developmental stages. An ontogenetic decrease in olfactory response, as

measured by EOG, was observed as tadpoles progress through prometamorphosis to

metamorphic climax (Fig. 4). Similarly, we observed a decrease in behavioural preference for

Spirulina between early prometamorphic tadpoles and older tadpoles closer to metamorphic

climax (Fig. 3). Previous studies have demonstrated that during the transition of the PC

olfactory epithelium, from the larval to the adult form, all OSNs suffer apoptosis and are

replaced (Higgs and Burd 2001; Dittrich et al. 2016). In X. laevis, the highest rate of OSN

apoptosis coincides with the maximum concentrations of plasma THs during metamorphic

climax (Dittrich et al. 2016). In L. catesbeiana, THs increase to their maximal concentrations

after TK XVII (reviewed in White and Nicoll 1981). Therefore, the reduced olfactory responses

to Spirulina extract in the older, TK XVIII-XX tadpoles when compared to younger,

prometamorphic tadpoles may be attributed to the widespread apoptosis of OSNs in the larval PC

olfactory epithelium. Alternately, metamorphic climax is also the period where the PC prepares

to detect airborne odorants (Taniguchi et al. 1996; Hansen et al. 1998). Therefore, it is also

possible that OSNs in the tadpole PC are not tuned to detect aqueous Spirulina extract and L-

alanine stimuli at TK stage XVIII-XX. However, qualitative ontogenetic changes in the cellular

structure of the olfactory epithelium were not observed in the PC between prometamorphic L.

catesbeiana tadpoles and those approaching metamorphic climax. Mucus sometimes limited our

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surface investigation of the ontogeny of the olfactory epithelium (Fig. 1). Future use of TEM

and histology would allow for a more detailed picture of the ontogenetic changes in the olfactory

epithelium, including cell turnover.

Interestingly, there was a disparity between stage-specific responses to Spirulina extract

when considering behavioural and EOG measurements in late prometamorphic (TK XVII)

tadpoles and those approaching metamorphic climax (TK XVIII-XX). Measured EOG responses

represent the bulk depolarization of OSNs to which odour molecules have bound. Action

potentials are generated when OSNs depolarize to the threshold potential, then the signal is

propagated to the brain where it is processed and an appropriate behavioural response is returned

(Baldwin and Scholz 2005). However, EOG does not indicate if the depolarization leads to

propagation of an action potential to the brain. Instead, it only measures a bulk influx of cations

from the extracellular environment associated with neural depolarization. Regardless, early

prometamorphic tadpoles exhibited a significant attraction response to Spirulina extract (Fig.

3A), but this behavioural response was considerably more variable in late prometamorphic

tadpoles and absent in tadpoles approaching metamorphic climax (Fig. 3B, C). Our results

highlight an interesting correlation between the known cessation of feeding at metamorphic

climax (Hourdry et al. 1996), and an ontogenetic change in olfactory response.

In summary, the results of the present study demonstrate the ontogenetic changes in the

olfactory system in L. catesbeiana tadpoles as they progress through to metamorphic climax. It

is evident that the olfactory structures on the PC olfactory epithelium develop gradually. At

early stages of premetamorphosis, sensory structures are underdeveloped, and therefore EOG is

unsuccessful. In addition, tadpoles approaching metamorphic climax also exhibit olfactory-

based ontogenetic changes, with a decreased olfactory response when compared to early and late

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prometamorphic tadpoles. This decrease in olfactory response may be related to other changes

co-occurring during metamorphosis and should be further investigated. Our results also showed

differences in the timing of olfactory development and initiation of function in L. catesbeiana as

compared to previous work on X. laevis, highlighting the importance of assessing olfactory

ontogeny and function in a diversity of anuran species.

Acknowledgements

Thank you to Doug Bray for aiding with preparation of specimens for SEM and to Grant

Duke for the use of the SEM at the Lethbridge Research and Development Centre, Lethbridge,

AB. This work was financially supported by the Natural Sciences and Engineering Research

Council [grant number STPGP 447250] and a Campus Alberta Innovation Program (CAIP)

Chair in Aquatic Health to G.G.P.

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Tables

Table 1. Classification of Taylor and Kollros (TK; 1946) stages used to assign North American

bullfrog tadpoles (Lithobates catesbeiana Shaw 1802) into the developmental groups used in the

present study.

TK Stages Developmental Group

I - X Premetamorphic

XI - XV Early prometamorphic

XVII Late prometamorphic

XVIII - XX Approaching metamorphic climax

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Figure captions

Fig. 1. Representative scanning electron micrographs, with insets of higher magnification

showing key features, of the principle cavity olfactory epithelium of North American bullfrog

(Lithobates catesbeiana Shaw 1802) tadpoles and corresponding EOG responses to Spirulina

spp. Turpin Ex Gomont 1893 (algae) extract. A – B: Developmental stage TK V. Polygonal

cells (asterisks) are evident in the olfactory epithelium, and some appear domed. Short

microvilli (arrows) are also present. Insets show a higher magnification view of microvilli (A)

and polygonal cells (B). C: TK V. EOG responses to Spirulina extract are undetectable. D: TK

VI. Polygonal cells (asterisks) are evident with microvillous projections (inset). E: TK VI. In a

few individuals, extensive cilia originating from non-sensory cells (arrows and left inset bracket)

and mucus (asterisks and right inset) cover the olfactory epithelium. Olfactory knobs with

ciliated projections (see insets in G for higher magnification exemplars) are present in only a few

individuals. F: Lack of EOG response to Spirulina extract at TK VI. One TK VI individual did,

however, exhibit a variable EOG response to Spirulina extract. G: TK IX. Cilia (arrows) and

mucus (black asterisks) cover the olfactory epithelium. Olfactory knobs with ciliated projections

(insets; white asterisks) are present. H: TK XIII. Relative to earlier stages, the entire surface of

the olfactory epithelium is more densely covered with cilia (arrows and inset) and mucus

(asterisks) obscuring olfactory knobs. I: Beginning at TK IX, EOG measurements of responses

to Spirulina extract are successful. Scale bars: A, D, E, G = 30 μm, B = 40 μm, H = 50 μm. Inset

scale bars: A, D, and H = 7.5 μm, B = 10 μm, E = 5 µm (both), G = 4 μm (both). Dashed line in

C, F, and I indicate stimulus delivery. Faint black horizontal line in G is an artefact of the SEM

scan.

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Fig. 2. Time premetamorphic North American bullfrog (Lithobates catesbeiana Shaw 1802)

tadpoles (TK range I – VIII) spent in the blank (dechlorinated water) and stimulus (amino acid

mixture) arms of the linear trough-style choice maze with or without anosmia induced (means ±

SE). A: Response when tadpole olfactory pits were left intact (n = 10; paired Wilcoxon rank

sum t-test, p = 0.017. B: Response when tadpoles were rendered anosmic (n = 12; paired t-test, p

= 0.43). An asterisk above a bar denotes a significant difference (p ≤ 0.05).

Fig. 3. Time spent by North American bullfrog (Lithobates catesbeiana Shaw 1802) tadpoles

undergoing prometamorphosis and those approaching metamorphic climax in the blank

(dechlorinated water) and stimulus (Spirulina spp. Turpin Ex Gomont 1893 extract) arms of the

choice maze (means ± SE). A: Early prometamorphic tadpole response (TK XI – XII, n = 9;

paired t-test, p = 0.01). B: Late prometamorphic tadpole response (TK XVII, n = 7; paired

Wilcoxon Rank Sum test, p = 0.19). C: Response of tadpoles approaching metamorphic climax

(TK XVIII – XX, n = 9; paired t-test, p = 0.71). An asterisk above a bar denotes a significant

difference (p ≤ 0.05).

Fig. 4. Raw EOG responses (EOG traces) and blank-corrected EOG responses of North

American bullfrog (Lithobates catesbeiana Shaw 1802) prometamorphic tadpoles

(prometamorphic, n = 11) and tadpoles approaching metamorphic climax (metamorphic, n = 12)

(means ± SE). A: Response to Spirulina spp. Turpin Ex Gomont 1893 extract (independent

samples t-test, p = 0.02). B: Response to L-alanine (Wilcoxon Rank Sum test, p = 0.16). An

asterisk denotes a significant difference (p ≤ 0.05).

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Fig. 5. Scanning electron micrographs of the principle cavity olfactory epithelium of North

American bullfrog (Lithobates catesbeiana Shaw 1802) tadpoles in (A) late prometamorphosis

(TK XVII) and (B) approaching metamorphic climax (TK XVIII – XX). At both stages, dense

cilia (arrows) and mucus (asterisks) are present in abundance, forming a dense matrix over the

olfactory knobs as in TK XIII tadpoles (see Fig. 1H). Scale bars: A = 50 μm, B = 40 μm.

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Tables

Table 1. Classification of Taylor and Kollros (TK; 1946) stages used to assign North American

bullfrog tadpoles (Lithobates catesbeiana Shaw 1802) into the developmental groups used in the

present study.

TK Stages Developmental Group

I - X Premetamorphic

XI - XV Early prometamorphic

XVII Late prometamorphic

XVIII - XX Approaching metamorphic climax

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Fig. 1. Representative scanning electron micrographs, with insets of higher magnification showing key features, of the principle cavity olfactory epithelium of North American bullfrog (Lithobates catesbeiana

Shaw 1802) tadpoles and corresponding EOG responses to Spirulina spp. Turpin Ex Gomont 1893 (algae) extract. A – B: Developmental stage TK V. Polygonal cells (asterisks) are evident in the olfactory

epithelium, and some appear domed. Short microvilli (arrows) are also present. Insets show a higher magnification view of microvilli (A) and polygonal cells (B). C: TK V. EOG responses to Spirulina extract are undetectable. D: TK VI. Polygonal cells (asterisks) are evident with microvillous projections (inset). E: TK VI. In a few individuals, extensive cilia originating from non-sensory cells (arrows and left inset bracket)

and mucus (asterisks and right inset) cover the olfactory epithelium. Olfactory knobs with ciliated projections (see insets in G for higher magnification exemplars) are present in only a few individuals. F: Lack of EOG response to Spirulina extract at TK VI. One TK VI individual did, however, exhibit a variable

EOG response to Spirulina extract. G: TK IX. Cilia (arrows) and mucus (black asterisks) cover the olfactory epithelium. Olfactory knobs with ciliated projections (insets; white asterisks) are present. H: TK XIII.

Relative to earlier stages, the entire surface of the olfactory epithelium is more densely covered with cilia (arrows and inset) and mucus (asterisks) obscuring olfactory knobs. I: Beginning at TK IX, EOG

measurements of responses to Spirulina extract are successful. Scale bars: A, D, E, G = 30 μm, B = 40 μm, H = 50 μm. Inset scale bars: A, D, and H = 7.5 μm, B = 10 μm, E = 5 µm (both), G = 4 μm (both). Dashed line in C, F, and I indicate stimulus delivery. Faint black horizontal line in G is an artefact of the SEM scan.

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Fig. 2. Time premetamorphic North American bullfrog (Lithobates catesbeiana Shaw 1802) tadpoles (TK range I – VIII) spent in the blank (dechlorinated water) and stimulus (amino acid mixture) arms of the

linear trough-style choice maze with or without anosmia induced (means ± SE). A: Response when tadpole olfactory pits were left intact (n = 10; paired Wilcoxon rank sum t-test, p = 0.017. B: Response when tadpoles were rendered anosmic (n = 12; paired t-test, p = 0.43). An asterisk above a bar denotes a

significant difference (p ≤ 0.05).

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Fig. 3. Time spent by North American bullfrog (Lithobates catesbeiana Shaw 1802) tadpoles undergoing prometamorphosis and those approaching metamorphic climax in the blank (dechlorinated water) and

stimulus (Spirulina spp. Turpin Ex Gomont 1893 extract) arms of the choice maze (means ± SE). A: Early prometamorphic tadpole response (TK XI – XII, n = 9; paired t-test, p = 0.01). B: Late prometamorphic tadpole response (TK XVII, n = 7; paired Wilcoxon Rank Sum test, p = 0.19). C: Response of tadpoles

approaching metamorphic climax (TK XVIII – XX, n = 9; paired t-test, p = 0.71). An asterisk above a bar denotes a significant difference (p ≤ 0.05).

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Fig. 4. Raw EOG responses (EOG traces) and blank-corrected EOG responses of North American bullfrog (Lithobates catesbeiana Shaw 1802) prometamorphic tadpoles (prometamorphic, n = 11) and tadpoles approaching metamorphic climax (metamorphic, n = 12) (means ± SE). A: Response to Spirulina spp.

Turpin Ex Gomont 1893 extract (independent samples t-test, p = 0.02). B: Response to L-alanine (Wilcoxon Rank Sum test, p = 0.16). An asterisk denotes a significant difference (p ≤ 0.05).

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Fig. 5. Scanning electron micrographs of the principle cavity olfactory epithelium of North American bullfrog (Lithobates catesbeiana Shaw 1802) tadpoles in (A) late prometamorphosis (TK XVII) and (B) approaching

metamorphic climax (TK XVIII – XX). At both stages, dense cilia (arrows) and mucus (asterisks) are present in abundance, forming a dense matrix over the olfactory knobs as in TK XIII tadpoles (see Fig. 1H).

Scale bars: A = 50 μm, B = 40 μm.

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