BUILDING a RISK ASSESSMENT MODEL FOR MANAGEMENT OF PERSISTENT ENDODONTIC LESIONS

Copyright by
Kee Zhi Ling Michelle
2015


Abstract
Olfactory studies in zebrafish have provided enormous insights into how olfaction
occurs in an aquatic environment, triggering innate and stereotypical responses that
allow survival in the vast habitat. One of the odors detected by zebrafish is an alarm
substance from the lesioned skin of conspecifics, known as Schreckstoff. A feature of
the alarm substance is increased potency caused by heating, and glycan content. To
further characterize Schreckstoff, I have found that hyaluronan (HA), a simple linear
polysaccharide, which is broken to active signaling fragments by heat, evokes activity
in the olfactory system of larval zebrafish.

Through the use of wide-field

fluorescence microscopy to perform calcium imaging on transgenic zebrafish
expressing the genetically encoded calcium indicator GCaMP, I demonstrated that
HA is able to evoke calcium responses in the olfactory epithelium and bulb of
zebrafish as young as 5 days post fertilization. This suggests HA can also function as
an odor, in addition to its diverse size-dependent roles in cell signaling. Additionally,
I also describe and demonstrate the use of a microfluidic chip, as a “proof-ofconcept” to aid in characterization of the olfactory sensory neurons that are
responsible for detecting HA.

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Dedicated in loving memory of Yeye

“It doesn’t matter how long you take,
but learn from the experience and enjoy the process.”
Thank you Yeye, I finally did it.

v


Acknowledgement
This thesis would not be possible without the following people, to which my heartfelt
gratitude and appreciation cannot be expressed in mere written words:

To Associate Professor Suresh Jesuthasan – I thank you sincerely for your
continuous mentorship, patience, guidance and support since 2007, especially during
the course of my degree and my two pregnancies. Research aside, you will always
be the source of my motivation to seek maternal rights even as a PhD student.

To my thesis advisory committee members, Assistant Professor Marc Fivaz,
Assistant Professor Adam Claridge-Chang, Associate Professor William F.
Burkholder – My sincere thanks and gratitude for your invaluable knowledge, advice
and critical suggestions throughout these years.

To my colleagues in SJ lab - Thank you all for sharing your wealth of knowledge and
advice, in all aspects of science and life. Dr Ajay Mathuru, thank you for being my
“personal critic”. Special thanks to my fellow graduate student - Joanne, Lin Qian,
Charlie and Mahathi - for keeping me healthy and mentally alive with our daily “4 p.m
chocolate and apple time”. Thank you all members of SJ lab, for converting me to a
caffeine addict, without which I would probably not appreciate the close relationship
between coffee and science.

To the members of MSB lab, both past and present – Thank you all for sharing your

invaluable knowledge and advice on qPCR, microfluidics and RNA sequencing.

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Last but not the least, I would like to express my deepest gratitude and appreciation
for my dear family members, especially to my parents for finally supporting me in
neuroscience endeavors; my husband for never failing to listen to my thoughts and
providing me with a loving refuge from science; my lovely sons, for keeping me
grounded, sane and youthful in the lab when the going gets tough and inspiring me
to understand cognitive development, processing and functions in young children.

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Contents
Title Page ................................................................................................................... i
Abstract Signature ..................................................................................................... ii
Copyright .................................................................................................................. iii
Abstract .................................................................................................................... iv
Dedication ................................................................................................................. v
Acknowledgements .................................................................................................. vi
Table of Contents .................................................................................................... viii
List of Tables ............................................................................................................ ix
List of Figures ............................................................................................................ x
Chapter 1. Introduction .............................................................................................. 1
1.1 Difference between olfaction and gustation.................................................... 2
1.2 Olfaction ........................................................................................................ 3
1.2.1 How olfaction differs in terrestrial and aquatic animals .......................... 4
1.2.2 Olfaction begins in utero........................................................................ 5

1.2.3 Food, foraging and homing odors.......................................................... 7
1.2.4 Olfaction in social context...................................................................... 8
1.2.4.1 Recognizing individuals ................................................................ 8
1.2.4.2 Recognizing kin ............................................................................ 9
1.2.4.2.1 Kin recognition and mate choice – role of MHC ................. 10
1.2.4.2.2 Kin recognition and mate choice – role of major urinary
proteins.............................................................................. 11
1.2.4.3 Recognizing predators................................................................ 12
1.3 Recognizing danger – smell of threatening situations .................................. 13
1.3.1 Source of Schreckstoff ........................................................................ 14
1.3.2 Components of Schreckstoff ............................................................... 14
1.4 Hyaluronan .................................................................................................. 17
1.4.1 Introduction of hyaluronan ................................................................... 17
1.4.2 Role of HA depends on its polymer size .............................................. 18

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1.4.3 Role of HA depends on its binding proteins ......................................... 20
1.4.4 HA degradation ................................................................................... 21
1.4.4.1 Enzymatic fragmentation of HA ............................................ 21
1.4.4.2 Thermal fragmentation of HA ................................................ 22
1.4.4.3 Degradation of HA by free radicals ....................................... 23
1.5 Introduction to zebrafish olfactory system .................................................... 24
1.5.1 Advantages of zebrafish as a model system ....................................... 24
1.5.2 Anatomy of the zebrafish olfactory system .......................................... 25
1.5.3 Types of OSNs.................................................................................... 26
1.5.4 Olfactory bulb ...................................................................................... 30
1.5.4.1 Odor coding in the olfactory bulb .......................................... 32
1.5.5 Projection from OB to higher brain centers .......................................... 33

1.6 Aims for this thesis ....................................................................................... 36
Chapter 2. Zebrafish detect hyaluronan as an odor ................................................. 37
2.1 Abstract ....................................................................................................... 37
2.2 Introduction .................................................................................................. 37
2.3. Material and Methods ................................................................................. 40
2.3.1 Animals ............................................................................................... 40
2.3.2 Skin extract preparations..................................................................... 41
2.3.3 Calcium imaging experiments ............................................................. 41
2.3.4 Odors stimulation ................................................................................ 42
2.3.5 Calcium image processing and analysis.............................................. 43
2.3.6 Dissection of olfactory epithelia ........................................................... 44
2.3.7 Confocal imaging ................................................................................ 44
2.4 Results ........................................................................................................ 45
2.4.1 Experimental workflow ........................................................................ 45
2.4.2 Et(sqKR15-3A) labels olfactory sensory neurons ................................ 47
2.4.3 OSNs respond distinctly when exposed to separate odors .................. 50

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2.4.4 A component in the WGA column elution buffer activates calcium
activity in OSNs ........................................................................................... 56
2.4.5. Different sizes of HA activates the olfactory bulb ................................ 57
2.4.6 Distinct glomeruli can be identified with Tg(NBT:GCaMP5) ................. 60
2.4.7 Exposure to a range of HA sizes at similar concentrations displayed
distinct responses over time ......................................................................... 62
2.4.8 Nanomolar concentration range of HA-L inhibited medial anterior
glomeruli of the olfactory bulb ...................................................................... 65
2.4.9 Similar concentrations of HA-L and HA-M evoked activity at maG, mdG
and vmG clusters ......................................................................................... 69

2.4.10 Exposure to HA-S evoked excitatory activity specifically in mdG and
vmG clusters ................................................................................................ 71
2.4.11. HA evoked similar glomerular activities across sizes, which were
distinct from glomerular activities after L-lysine stimulation .......................... 75
2.4.12. Different sizes of HA at similar concentrations activated OSNs ........ 79
2.4.13 The olfactory bulb remains active to HA exposure after two trials...... 80
2.5. Discussion .................................................................................................. 83
Chapter 3. A microfluidic device to sort olfactory sensory neurons based on dynamic
response to different odors ...................................................................................... 95
3.1 Abstract ....................................................................................................... 95
3.2 Introduction .................................................................................................. 96
3.3. Material and Methods ................................................................................. 98
3.3.1 Microfluidic device fabrication ............................................................. 98
3.3.2 Isolation and dissociation of olfactory epithelial cells ........................... 98
3.3.3 Cell viability assay ............................................................................... 99
3.3.4 Calcium indicator dyes ........................................................................ 99
3.3.5 Ionophore and odorants stimulation .................................................. 100
3.3.6 Image acquisition and processing ..................................................... 100
3.3.7 qPCR of single OSN ......................................................................... 101
3.4 Results ...................................................................................................... 102
3.4.1 Microfluidic device design ................................................................. 102

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3.4.2 Device operation and preparation ..................................................... 103
3.4.3 Olfactory epithelial (OE) cells viability and sorting efficiency ............. 106
3.4.4 Microfluidic chip allows active monitoring of intracellular calcium
changes in response to external stimulus .................................................. 108
3.4.5. Microfluidic chip allows correct sorting and recovery of subpopulation of

cells ........................................................................................................... 110
3.5. Discussion ................................................................................................ 114
Chapter 4: References .......................................................................................... 117
Appendix A ............................................................................................................ 135
Biography .............................................................................................................. 144

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List of Tables
Table 2-1: Table of results for two-way ANOVA with Tukey’s multiple comparisons
post-hoc test ............................................................................................................ 78
Table 3-1: Cell sorting efficiency and recovery from different input conditions. ...... 107

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List of Figures
Figure 1-1: Schmatic representation of GAGs ......................................................... 19
Figure 1-2: Olfactory system of zebrafish ................................................................ 27
Figure 1-3: Four unique classes of olfactory sensory neurons (OSNs). ................... 29
Figure 1-4: Organization of the olfactory bulb network ............................................. 31
Figure 1-5: Schematic overview of axon projections from the olfactory bulb to other
forebrain regions. ................................................................................................... 35
Figure 2-1: Schematic diagram of experimental workflow ........................................ 46
Figure 2-2: Double fluorescent labeling of Et(sqKR15-3A)/NBT:GCaMP5 transgenic
zebrafish.................................................................................................................. 49
Figure 2-3 (A-F): Calcium activity in Et(sqKR15-3A)/NBT:GCaMP3 olfactory
epithelium at dorsal (A-F) planes ............................................................................. 53
Figure 2-3 (G-L): Calcium activity in Et(sqKR15-3A)/NBT:GCaMP3 olfactory

epithelium at ventral (G-L) planes ............................................................................ 54
Figure 2-4: Thunder analyses of Et(sqKR15-3A)/NBT:GCaMP3 olfactory epithelium
on exposure to various odors at dorsal and ventral planes. .................................... 55
Figure 2-5: Changes in calcium fluorescence intensity of L-lysine and different sizes
of HA with similar concentrations in olfactory bulb of NBT:GCaMP5. ...................... 59
Figure 2-6: Distinct glomeruli identified in olfactory bulb of Tg(NBT:GCaMP5). ....... 61
Figure 2-7: Thunder analyses of different sizes of HA with similar concentrations in
olfactory bulb of Et(sqKR15-3A)/NBT:GCaMP5. ..................................................... 64
Figure 2-8: Thunder analyses of lower concentrations of HA-L and L-lysine in
olfactory bulb of Tg(OMP:lyn-mRFP)/NBT:GCaMP5 ............................................... 67
Figure 2-9: Thunder analyses of higher concentrations of HA-L and L-lysine in
olfactory bulb of Tg(OMP:lyn-mRFP)/NBT:GCaMP5 ............................................... 68
Figure 2-10: Thunder analyses of HA-L and HA-M at similar concentrations, with Llysine in olfactory bulb of Tg(OMP:lyn-mRFP)/NBT:GCaMP5 .................................. 70
Figure 2-11: Thunder analyses of HA-M and HA-S at similar concentrations, with Llysine in olfactory bulb of Et(sqKR15-3A)/NBT:GCaMP5. ....................................... 73
Figure 2-12: Thunder analyses of HA-M and HA-S at a ten-fold higher concentration,
with L-lysine in OB of Et(sqKR15-3A)/NBT:GCaMP5. ............................................. 74
Figure 2-13: Changes in calcium fluorescence intensity of L-lysine and different sizes
of HA with similar concentrations in OSNs of Et(sqKR15-3A)/NBT:GCaMP3........... 77
Figure 2-14: HA evoked distinct glomerular activities from L-lysine stimulation in
various glomeruli clusters ........................................................................................ 81

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Figure 2-15: Et(sqKR15-3A)/NBT:GCaMP5 OB responses to two trials of various
stimuli. .................................................................................................................... 82
Figure 2-16. Mass spectrometry peak of active fraction of Schreckstoff .................. 89
Figure 2-17: Juvenile zebrafish significantly swam slower and closer to the wall after
250 nM-1µM HA-M delivery than when Schreckstoff was delivered ......................... 94
Figure 3-1: Overview of experimental design and diagram of microfluidic device…104

Figure 3-2: Operational characteristics for odorant stimulation and cell recovery ... 105
Figure 3-3: Ionophore A23187-induced calcium influx in zebrafish olfactory epithelial
cells ....................................................................................................................... 109
Figure 3-4: Lysine stimulation of a heterogenous cell population from OE of
TrpC2:gapVenus transgenic zebrafish ................................................................... 112
Figure 3-5: qPCR analyses of cells isolated after exposure to various odorants. .. 113

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Chapter 1: Introduction
"Smell is a potent wizard that transports us across thousands of miles and all the
years we have lived. The odors of fruits waft me to my southern home, to my
childhood frolics in the peach orchard. Other odors, instantaneous and fleeting,
cause my heart to dilate joyously or contract with remembered grief. Even as I think
of smells, my nose is full of scents that start to awake sweet memories of summers
gone and ripening fields far away."
-

Helen Keller

Imagine you are walking down an empty street on a cool early morning, with rays of
sunlight shining through the leaves of tall trees. You take a deep breath of fresh crisp
air, enjoying the moment as you hold your tumbler of freshly brewed coffee. Without
realizing it, we are surrounded by a treasure of sights, sounds, smells, and textures
that arouse our various sensory modalities, guiding our behavior in ways that we are
not conscious of. These modalities are vital for survival; without them, we would not
be able to select our food, communicate and navigate through our environment.
These sensory systems may even determine life or death. Animals use sensory cues
to explore the environment and retrieve vital information that are integrated and

processed in the brain, consequently allowing animals to respond in an adaptive
manner to enhance survival. Of these sensory cues, chemosensory communication
is phylogenetically the oldest form of communication between organisms. It is
present in organisms across phyla, from bacteria (Berg, 1975) to nematodes
(O’Halloran et al., 2006), arthropods (Rittschof, 1992) and mammals (Johnston et al.,
1993; Meyerhof and Korsching, 2009), discriminating other organisms from their
conspecifics and biological status through the release of specific chemicals into the
local surroundings.

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1.1.

Differences between olfaction and gustation

Chemosensory communication is deeply intriguing, as it functions in all environs (air,
land and sea) and continues to persist in spite of rapid evolutionary change
(Symonds and Elgar, 2008). While chemosensation includes chemethesis (pain,
touch and thermal dermal sensation), olfaction (smell) and gustation (taste) (Finger
et al., 2000), olfaction and gustation are nowadays identified as the most ubiquitous
of all sensory systems (Hildebrand and Shepherd, 1997; Ache and Young, 2005;
Penn, 2006). Furthermore, these two chemosensory systems are the most
perceptually intertwined: in humans, as presumably in other animals, the flavor of a
nutriment is highly regulated by the simultaneous perception of its odor.

In vertebrates, olfaction is defined as the detection chemicals at a long-range
distance, either in air or water, by olfactory sensory neurons (OSNs) with axons in
the olfactory nerve (cranial nerve I) (Hildebrand and Shepherd, 1997). Gustation,
however, requires direct contact with chemical compounds dissolved in solution and

is mediated by non-neuronal, polarized epithelial cells known as taste receptor cells
(TRCs). Additionally, TRCs are innervated by the facial (cranial nerve VII),
glossopharyngeal (cranial nerve IX) and vagal (cranial nerve X) nerves that project to
different brain structures and have diverse functions (Atema, 1977; Caprio et al.,
1993).

Studies underlying the molecular biology and neurobiology of sense of taste and
smell have also revealed fundamental differences in stimulus processing by these
two sensory systems. Olfactory circuits are optimized for combinatorial detection of a
vast number of odorants (Friedrich and Korsching, 1997; Malnic et al., 1999;
Araneda et al., 2000; Katada et al., 2005; Nakagawa et al., 2005; Hallem and
Carlson, 2006), while the gustatory system categorize taste into unique and

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topographically defined modalities – bitter, sour, salty, sweet and umami (the savory
taste of monosodium glutatmate) (Nelson et al., 2001; Zhang et al., 2003; Zhao et al.,
2003; Chandrashekar et al., 2006; Huang et al., 2006; Behrens and Meyerhof, 2009;
Chen et al., 2011).

Lastly, olfaction and gustation can be distinguished by their function. Gustation is
known to mediate simple, immediate reflexive behaviors, especially toward food
(such as choice and ingestion). Olfaction, on the other hand, tends to mediate more
complex behaviors, such as in mate selection, and sensing for predators (Todd et al.,
1967; Brechbühl et al., 2008; Gerlach et al., 2008; Getz and Page, 2010).

Although both olfaction and gustation are chemosensory systems, olfaction is the
principal chemosensory system used in most animals. Previously ranked as one of
the “lower” senses by ancient philosophers such as Plato and Aristotle, the olfactory

system remained the most enigmatic of our senses until 1991, when Linda Buck and
Richard Axel published a joint groundbreaking paper that sheds light on olfactory
receptors and how the brain recognizes and remembers a plethora of odors (Buck
and Axel, 1991). They were awarded the 2004 Nobel Prize in Physiology or Medicine
for their pioneering work that permits a deeper understanding of the olfactory system.
In this dissertation, the focus will mainly be on olfaction.

1.2.

Olfaction

Olfaction, the sense of smell, has baffled and also thrilled epistemologists since
philosophy began (Ackerman, 1990). It is known for its chemical sensitivity, with only
a few molecules of an odorant needed to cross a large distance before recognition,
awareness and distinction of the odor takes place with great precision. Yet, pure

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odors are rare in nature; odorants are commonly an array of chemical molecules.
Hence, as aptly highlighted by Bargmann, the olfactory system, unlike the visual and
auditory system which detects “immutable physical entities”, is analogous to the
immune system, with its “remarkable ability to track constantly changing cues
generated by other organisms, and continually generate, test and discard receptor
genes and coding strategies over evolution” (Bargmann, 2006).

1.2.1. How olfaction differs between terrestrial and aquatic animals

To further appreciate the complexity of olfaction, we must first understand and
distinguish how olfaction takes place in various animals and its many roles in

ensuring survival.

In the olfactory environment of terrestrial organisms, odors of distant objects are
brought to the nose through diffusion of odorant molecules by wind. These molecules
leave their source into the air, which itself contains other air packets of odorant
molecules from diverse sources. The airborne, volatile chemical molecules are then
mixed, as they move together with the wind. Consequently, odors that reach the
nose are composed of multi-molecular mixtures, often containing hundreds of volatile
components (Knudsen et al., 1993). Added complexity arises as the precise
composition of the odor is progressively mixed with other odors in the environment
over time (Bossert and Wilson, 1963; Wright, 1982; Murlis, 1992). Moreover, the odor
source changes during its lifetime, causing variations to the composition of an odor
due to processes such as oxidation (Maqsood and Benjakul, 2011).

4


Conversely, odors in aquatic environment are soluble chemicals in water. Despite not
encountering airborne volatile odorants, aquatic organisms possess olfactory
systems that are anatomically similar to terrestrial animals (Hildebrand and Shepherd,
1997; Laurent, 2002). Since chemicals cues move more slowly in water than in air,
most aquatic organisms have their OSNs positioned anteriorly, where they will be
exposed to moving water, allowing the organism to have a better chance to smell
and detect chemical changes in the environment. These chemical cues also form a
plume, which is well conserved at great distances from the source (Murlis, 1992).
Additionally, the ability to detect chemical cues over great distances is of particular
importance and advantage to aquatic animals due to limitations on vision at depth
and in complex or turbid environments.

1.2.2. Olfaction begins in utero

How early in life does olfaction begin? Ontogenetically, the olfactory system is
developed closely with the somesthesic and vestibular modalities (Gottlieb, 1971a). It
is fully functional during gestation, preceding the development of both visual and
auditory systems (Gottlieb, 1971b). Despite this fact, olfaction in utero was not
considered seriously as early researchers assumed the necessity for presence of
airborne chemical molecules and fluid currents flowing through the fetus’ nose to
trigger olfaction (Humphrey, 1978). However, later advancements in olfactory system
research changed this notion. Teicher and Blass (1977) first provided the initial
evidence that newborn albino rats are attracted to amniotic fluid and that the olfactory
cues contained in it guide the initial sucking episode when auditory and visual
systems are not fully developed. Subsequent studies also showed that neonates are
highly responsive and had more specific preference to maternal biological odors,

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which they have never been exposed to prior to the post-natal experience. These
odor preferences can be acquired within a short time window after birth (Sullivan and
Wilson, 1991; Kindermann et al., 1994) and include their own mothers’ saliva
(Teicher and Blass, 1977), breast milk and amniotic fluid in mammals such as
rodents (Smotherman, 1982; Hepper, 1987; Smotherman and Robinson, 1990),
piglets (Parfet and Gonyou, 1991), sheep (Schaal et al., 1995) and humans (Varendi
et al., 1996; Schaal et al., 1998, 2000). From these studies, the most obvious way to
stimulate fetal olfactory receptors is through the amniotic fluid, either through direct
infusion or maternal ingestion. In fact, the human fetus inhales more than twice the
volume it swallows (Duenhoelter and Pritchard, 1976), suggesting an intense
movement of fluid through the nose, thus allowing olfaction in utero. The chemical
composition in the amniotic fluid at a specific period of fetal development may also
induce a specific pattern of olfactory receptor expression, therefore determining their

odorant specificity and sensitivity (Wang et al., 1993).

Along with the ability to detect, learn and retrieve odor information from the late
gestation period, the fetal chemoreceptor system is also genetically predisposed to
detect particular biological odor cues (Todrank et al., 2005). These are essential for
neonatal survival as the fetus prepares itself for life outside the womb, by locating
food, eating and attaching to its biological mother for protection during development
(Schaal et al., 1998). However, the neonate does not solely determine the motherinfant interaction. In humans, mothers also have the capacity to distinguish between
the smell of their infant and other children (Porter et al., 1983). The importance of
olfaction for establishing maternal behavior is also observed in ewes (Lévy et al.,
1996). Aminotic fluids, generally repulsive, briefly become attractive at parturition
(Lévy et al., 1996). In contrast, the lack of amniotic fluids in neonates, as well as
suppression of olfactory cues is detrimental for maternal acceptance, leading to an

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absence of licking, refusal to nurse and aggressive maternal behavior (Lévy et al.,
1996).

1.2.3. Food, foraging and homing odors

Olfaction in utero is also responsible for allowing one to recognize whether food is fit
for ingestion. Future food preferences are influenced by odors perceived in the womb
as well. Several experimental paradigms have demonstrated neonatal rats and
humans can associate chemosensory stimulation experienced in the womb with
aversive (Smotherman, 1982) or appetitive (Pedersen et al., 1983; Schaal et al.,
1995, 2000) reinforcements that may influence their behavior throughout their lifetime.
Reversible changes in food odor preferences can also occur over short periods
because of simple exposure, in a phenomenon known as sensory-specific satiety

(Rolls et al., 1981). In general, responsiveness and attraction towards certain food
odors are modified by hunger and specific feeding history - one that is fed to satiety
on a particular food, may have decreased attraction to it and continue to seek other
foods to satisfy their hunger.

In aquatic animals, olfactory information from a distance may also elicit foraging
behavior as with lobsters (Panulirus interruptus), which have a marked specific
preference for abalone and mackerel muscle that are presented from one to two
meters apart (Zimmer-Faust and Case, 1982).

Additionally, one of the classic functions of olfaction in aquatic animals is for homing
and home range recognition, as observed in salmon (Oncorhynchus spp.), which
return to their natal stream to spawn after spending years in the Pacific Ocean based
on learned odor cues (Dittman and Quinn, 1996). Salmon first rendered anosmic by

7


plugging their nostrils and were recaptured evenly among all streams of the basin,
whereas salmon with unplugged noses had higher correlation of returning to home
stream.

1.2.4. Olfaction in social context
1.2.4.1.

Recognizing individuals

Olfaction also plays a critical role in the animals’ social behavior. As individuals are
fundamental units of social interaction and organization, the abilities to discriminate
and recognize individuals are crucial in understanding social behavior. Odors from

the same animal may provide diverse information about the animal, as demonstrated
in mammals. Castroreum secreted by beavers is involved in territorial demarcation
and may mediate recognition of family members, whereas anal gland secretion
contains individual, kin and sex information (Sun and Müller-Schwarze, 1999). On
the other hand, odor cues from the same animal may provide redundant information.
For example, in golden hamsters, five different odor sources were individually
distinctive, namely flank gland, ear glands, urine, feces and vaginal secretions,
where other sources of odors were not (Johnston et al., 1993). In three-spined
sticklebacks (Gasterosteus aculeatus), the odor of an individual is strongly influenced
by both recent habitat use and diet (Ward, 2004). From wasps to fish, rodents and
primates, this odor information is a consequence of varying proportions of individual
chemical compounds in a complex mixtures of chemicals, producing a distinct odor
gestalt that is easily recognized by individuals of the same species (Penn and Potts,
1998a; Dani et al., 2001; Smith et al., 2001; Leinders-Zufall et al., 2004; Ward, 2004;
Hinz et al., 2013).

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1.2.4.2.

Recognizing kin

Olfaction is also fundamental in kin recognition as it facilitates cooperation among
relatives, avoiding excessive kin competition or inbreeding (Hamilton, 1964).
Individuals with the advantage of discerning a kin member could have had higher
levels of survival, successful reproduction and consequently higher fitness. At least
two mechanisms have been demonstrated to recognize kins through olfaction: by
self-inspection (also known as “self-referent phenotype matching” and the “armpit
effect”) or indirect familiarity (“phenotype matching”).

The “armpit effect” is defined as an individual compares its own phenotype (such as
odors) to that of other conspecifics . The other conspecific would be treated as kin if
there is a high similarity to odor of self. Such example has been shown in female
golden hamsters. Through cross-fostering studies shortly after birth, estrous females
were more attracted to unfamiliar non-kin than to unfamiliar kin (Mateo and Johnston,
2000).

In phenotype matching, individuals learn, recognize and associate characteristics of
other conspecifics they grow up with instead. Tang-Martinez showed that vertebrates
who resemble their own kin later in life are treated as related (Tang-Martinez, 2001).
Aquatic vertebrates such as juvenile zebrafish (Gerlach and Lysiak, 2006) and cichild
fish (Pelvicachromis taeniatus) (Hesse et al., 2012) have demonstrated to use
phenotype matching based on olfactory cues to differentiate between kin and non-kin,
preferring odor of unfamiliar siblings to unfamiliar and unrelated individuals. This
leads to an immediate selective advantage as juvenile fishes housed in kin groups
grew significantly faster than those in groups of unrelated individuals (Gerlach et al.,
2007). Wild Atlantic salmon (Salmo salar L.) that have an accelerated growth

9


frequently correlates with enhanced survival and earlier reproductive age (Garant et
al., 2003).

As summarized by Krause and Krüger (2012), the finding that even zebrafinches
(Taeniopygia guttata), previously known for recognizing relatives through auditory
signals, can rely on olfactory cues for kin recognition, leads to the conclusion that
general mechanism of kin recognition might be based on phenotype matching
through genetically-based markers, such as the major histocompatibility complex

(MHC). A specific set of MHC peptides have been recently found to play a novel role
in olfactory imprinting of kin in a specific line of zebrafish, activating specific
populations of neurons in the olfactory bulb that do not overlap with food odor (Hinz
et al., 2013).

1.2.4.2.1.

Kin recognition and mate choice – role of MHC

MHC is one of the two key polymorphic and multigenic complexes found in odors that
contribute to the determination of individual differences. Known to play a central role
in immunological self/non-self-recognition (Janeway et al., 2001), MHC genes are
characterized by their high polymorphism, making MHC similarity between
individuals a good indicator for their relatedness. House mice have shown preference
to mate with individuals carrying dissimilar MHC genes under laboratory (Eklund,
1997; Carroll et al., 2002) and semi-natural conditions (Potts et al., 1991). Female
mice in estrus show preferences towards odors of males with a dissimilar MHC type
when tested in a Y-maze, whereas those not in estrus did not show such preference
(Egid and Brown, 1989). Similarly, women in the fertile phase of their menstrual cycle
are most attracted to the odor of MHC-dissimilar individuals (Wedekind and Füri,
1997) and have mating preferences towards these individuals (Ober et al., 1997).

10


Such preferences are reversed when women are taking oral contraceptives – odors
of men with a similar MHC type are then preferred (Roberts et al., 2008).

These selective behaviors reduce inbreeding (Penn and Potts, 1998a) and enhance
MHC variability and individual odor distinctiveness, as mating with a male of

dissimilar MHC type produces offspring that are more heterozygous across all of the
genome, not just at the MHC loci. These offspring also respond effectively to a wider
range of pathogens than homozygous pups (Penn and Potts, 1998b), hence
increasing their ability to survive. Similarly, females of three-spined sticklebacks
(Gasterosteus aculeatus) used an odor-based selection strategy to achieve optimal
number of MHC alleles in their offspring, equipping them with optimal resistance
toward pathogens and parasites. When gravid female sticklebacks are exposed to
sources of water from two males, females appear to compare their own set of MHC
alleles and show preferences for the scent of the male with the optimal complement
of alleles (Reusch et al., 2001; Milinski et al., 2005).

1.2.4.2.2.

Kin recognition and mate choice - role of major urinary proteins

In contrast, a recent study using wild mice in large enclosures has shown that MHC
is not a relevant marker that animals use for avoiding inbreeding (Sherborne et al.,
2007). Instead, major urinary proteins (MUPs) are the main responsible markers,
containing individually distinctive odor information (Hurst et al., 2001; Cheetham et
al., 2007). MUPs are mostly produced in the liver and become concentrated in the
urine of mice, though similar lipocalin proteins in scent-producing organs of other
species have been shown in other rodents (Hurst et al., 2001; Beynon and Hurst,
2004). Although they are non-volatile molecules, they bind to smaller volatile

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