REVIEW

Behavioural and neuronal basis of olfactory imprinting and kinrecognition in larval fishGabriele Gerlach1,2,3,*, Kristin Tietje1, Daniela Biechl4, Iori Namekawa5, Gregor Schalm1 and Astrid Sulmann1

ABSTRACTImprinting is a specific form of long-term memory of a cue acquiredduring a sensitive phase of development. To ensure that organismsmemorize the right cue, the learning process must happen during aspecific short time period, mostly soon after hatching, which shouldend before irrelevant or misleading signals are encountered. A well-known case of olfactory imprinting in the aquatic environment isthat of the anadromous Atlantic and Pacific salmon, which prefer theolfactory cues of natal rivers to which they return after migratingseveral years in the open ocean. Recent research has shown thatolfactory imprinting and olfactory guided navigation in the marinerealm are far more common than previously assumed. Here, wepresent evidence for the involvement of olfactory imprinting in thenavigation behaviour of coral reef fish, which prefer their home reefodour over that of other reefs. Two main olfactory imprintingprocesses can be differentiated: (1) imprinting on environmentalcues and (2) imprinting on chemical compounds released by kin,which is based on genetic relatedness among conspecifics.While thefirst process allows for plasticity, so that organisms can imprint on avariety of chemical signals, the latter seems to be restricted to specificgenetically determined kin signals. We focus on the second,elucidating the behavioural and neuronal basis of the imprintingprocess on kin cues using larval zebrafish (Danio rerio) as a model.Our data suggest that the process of imprinting is not confined to thecentral nervous system but also triggers some changes in theolfactory epithelium.

KEY WORDS: Coral reef fish, Larval dispersal, Orientation, MHCpeptides, Olfaction, Crypt cell

IntroductionOlfactory guided navigation in coral reef fishLike many aquatic organisms, coral reef fish show a dual life stage,where settled adults produce dispersing larvae. At hatching,planktonic larvae drift away from the reef to spend a species-dependent time (larval dispersal duration) in the open ocean –probably to avoid high predation in the reef. These millimetre-sizedlarvae quickly (within a few days of dispersal) turn into relativelypowerful juvenile swimmers, reaching swimming speeds of severalcentimetres per second (Fisher et al., 2000; Fisher and Wilson,

2004; Stobutzki and Bellwood, 1994, 1997). Until recently, thedistribution and settlement of coral reef fish were assumed to bepurely driven by currents and stochastic storm events. However, thepersistence of marine populations at small isolated oceanic islandsrequires that a significant number of juveniles return to the natalhabitat after their pelagic dispersal phase (Robertson, 2001). Here,and even in less isolated habitats, self-recruitment and natal hominghas been assumed to be (far) greater than purely planktonicdispersal would predict from modelling approaches (Armsworth,2000; Cowen et al., 2000; Staaterman and Paris, 2014; Wolanskiet al., 1997). We used population genetic analysis to demonstratethat up to 60% of juvenile cardinalfish, Ostorhinchus doederleini,could be assigned to the adult reef population where they wereabout to settle (Gerlach et al., 2007a, 2016). Natal homing ofother species, e.g. clownfish juveniles (Amphiprion spp.) wasalso confirmed by mark–recapture studies, otolith tagging andmicrochemistry studies (Jones et al., 1999; Swearer et al., 1999;Thorrold et al., 2006).

As it is not possible to track larvae in the ocean, dispersaldistances are based on catching larvae in plankton tows and bymodelling approaches using the pelagic larval duration as a proxy(but see Weersing and Toonen, 2009). Dispersal distances of coralreef fish larvae are assumed to be shorter than 150 km depending onthe species (Burgess et al., 2007; Paris and Cowen, 2004). Despite apotentially wide distribution, numbers of homing coral reef fishjuveniles are far higher than expected by random movement.Orientation capabilities could help them to find their way back totheir natal reefs.

Finding the way back to a natal reef, river or beach after week- oryear-long dispersal might require a learning and memory processinvolving time-dependent specific parameters of this natal place.For orientation-guided homing to natal reefs, coral reef fish larvaemust remember sensory parameters that they experienced directlyafter hatching, which is the only time in which they can obtainreliable ‘home cues’, as larvae – with fully developed olfactory andvisual sensory systems – start dispersing into the open ocean on thenight of hatching.

To explain navigation over long distances, we provided evidencethat the juvenile cardinalfish (O. doederleini) can use time-compensated sun compass orientation during the day (Mouritsenet al., 2013) and a magnetic compass at night (Bottesch et al., 2016).For orientation at closer distances, there is evidence that reef fishlarvae can also respond to acoustic cues for orientation (Radfordet al., 2010, 2011). Additionally, we have documented apronounced importance of olfaction in the homing of coral reeffish larvae (Atema et al., 2002; Gerlach et al., 2007a; Paris et al.,2013). Coral reef fishes such as O. doederleini, Pomacentruscoelestis and several other species of apogonid and pomacentridjuveniles were shown to prefer the smell of water collected from areef to that of open ocean water (Atema et al., 2002). Both specieswere capable of distinguishing between chemical cues from reefs

1Institute of Biology and Environmental Sciences, Carl von Ossietzky UniversityOldenburg, 26129 Oldenburg, Germany. 2Helmholtz Institute for Functional MarineBiodiversity Oldenburg (HIFMB), 26129 Oldenburg, Germany. 3Centre ofExcellence for Coral Reef Studies and School of Marine and Tropical Biology,James Cook University, QLD 4811, Australia. 4Graduate School of SystemicNeurosciences & Department Biology II, Ludwig-Maximilians-Universitat Munich,82152 Planegg-Martinsried, Germany. 5Friedrich Miescher Institute for BiomedicalResearch, 4058 Basel, Switzerland.

*Author for correspondence (gabriele.gerlach@uni-oldenburg.de)

G.G., 0000-0001-5246-944X; I.N., 0000-0002-1012-9823

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within the Capricorn/Bunker reef group and preferred the smell ofwater from the reef where they were caught over that of other nearbyreefs (Gerlach et al., 2007a). We further demonstrated that thepreference for home reef odour did not switch even when larvaewere exposed to other reef odours for several days (Miller-Simset al., 2011). Therefore, we concluded that coral reef fish larvae hadimprinted on olfactory cues of their home reefs, similar to the well-known olfactory imprinting of salmon on chemical cues of natalrivers (Hasler et al., 1978; Scholz et al., 1973, 1976). However, therobust experimental manipulations that demonstrate an ability ofreef fish to imprint upon artificial odours in a similar way tosalmonids have yet to be performed.

Olfactory imprinting and olfactory guided navigation in teleostsImprinting is a specific form of long-term memory enablingorganisms to recognize specific cues from the environment andconspecifics. It might enable offspring to find their mothers amongmany other females or help animals to remember and recognizeenvironmental conditions that they experienced shortly afterhatching or birth. To ensure that organisms memorize the rightcue, the learning process must happen during a specific short timeperiod, which should end before the organism encounters irrelevantsignals from unrelated conspecifics or environments. One well-known example of a visual imprinting process is Konrad Lorenz’sgeese experiment (Lorenz, 1935) in which he showed that goslingsimprint on the first moving object that they experience afterhatching, which they take to be their mother. Many imprintingprocesses are involving olfactory cues, probably because in socialcontexts chemical cues can be taken as reliable ‘honest’ signals andmanipulating one’s own body smell is very difficult.The timing of imprinting has been studied extensively in

salmonids, and there is strong evidence that salmon imprint onenvironmental cues of their natal water during the parr–smolttransformation (PST) when they adapt from living in freshwater toliving in seawater (for review, see Dittman and Quinn, 1996). Thereis more recent evidence that some species of Pacific salmon, e.g.juvenile pink salmon (Oncorhynchus gorbuscha) and others, canimprint prior to the PST at a very young age (Bett et al., 2016;Dittman et al., 2015). Bett et al. (2016) published an excellentreview on olfactory imprinting and olfactory guided navigationduring spawning migrations in different salmon species andlampreys, elucidating imprinting cues and underlyingmechanisms. However, there is also evidence that salmon imprintmagnetically on their home area and this most likely brings themclose to the home stream, with olfactory cues used mainly in thefinal part of the spawning migration (Putman et al., 2014, 2013).In addition to natal water, anadromous fish also respond to water

that is conditioned by conspecifics, which they remember andrecognize years later, when they search for their natal river (forreview, see Keefer and Caudill, 2014). There is evidence thatsalmonids are even capable of population-level discrimination(Courtenay et al., 1997; Doving et al., 1974; Groot et al., 1986;McBride et al., 1964; Nordeng, 1971; Nordeng and Bratland, 2006;Olsén, 1989; Quinn and Tolson, 1986; Selset and Doving, 1980),and even discrimination of siblings from non-siblings (Quinn andBusack, 1985; Quinn and Hara, 1986; Winberg and Ols, 1992). Butsuch kin-structured associations are not confined to salmonids.Genetic analyses have shown that in numerous vertebrate andinvertebrate species, populations do not consist of a random mix ofindividuals but of groups of related individuals of full- or half-siblings; kin associations have been demonstrated in many teleostspecies (for review, see Gerlach and Hinz, 2012; Selwyn et al.,

2016). To identify even unfamiliar kin, organisms must imprint onspecific cues of kin, which they match with those expressed byunknown conspecifics (phenotype matching) (Hepper, 1986; Tang-Martinez, 2001). Therefore, our findings (Gerlach et al., 2008;Gerlach and Lysiak, 2006; Hinz et al., 2013a,b) on the mechanismsunderpinning olfactory imprinting in zebrafish (Danio rerio) mightapply to a whole variety of species.

Behavioural basis of olfactory imprintingOlfactory imprinting on conspecific and kin cues in zebrafishWhile selective advantages of kin recognition were studied innumerous species, the behavioural and neurobiological basis of theimprinting process are much less understood. To elucidate thebehavioural and neuronal basis of the imprinting process, we tookadvantage of the model organism zebrafish (D. rerio), whichimprint on an olfactory and visual template of kin early indevelopment, leading to a long-term memory expressed as anolfactory-based differentiation between kin and non-kin (Hinz et al.,2013a).

Zebrafish raised in kin groups (siblings) identify even unfamiliarkin later in life, a process called ‘phenotype matching’ (Hepper,1986). We found that larval and juvenile zebrafish preferredolfactory cues of kin when they were given the choice betweenwater in which kin (siblings) or non-kin were kept; larvae weretested in a two-channel ‘Atema’ choice flume (Mann et al., 2003).The olfactory preference for kin changed with sexual maturity: adultfemales preferred the odour blend of unfamiliar non-related malesover unfamiliar brothers, while adult males were attracted to allfemales, related and unrelated (Gerlach and Lysiak, 2006).

We raised larvae under different conditions to identify the timewindow of imprinting (see Fig. 1F). As experimentally isolatedlarvae (kept in glass beakers) did not express any olfactorypreference in the Atema choice flume test, we concluded that theymust be exposed to siblings during development. The sensitiveperiod for olfactory templates of kin is limited to 24 h at 6 days post-fertilization (dpf ) (Gerlach et al., 2008) (Fig. 1A).When larvaewereexposed to kin at other days for 24 h, but not at 6 dpf, they did notexpress any kin preference later in life (Fig. 1F). We also raisedsingle larvae (hatching occurs at 4–5 dpf at a temperature of 25°C)in separate beakers and could imprint themwhen adding kin water at6 dpf (Fig. 1B). Without such olfactory cues, they did not expressany preference for kin (Fig. 1C,E). Interestingly, the application ofnon-kin water at 6 dpf did not result in a preference for non-kinodour (Fig. 1G). Therefore, zebrafish larvae must experience kinodour at 6 dpf for imprinting, and they cannot be imprinted onolfactory cues of non-kin.

Placing the glass beakers containing single larvae directly next toeach other was very important, because it turned out that a visualtemplatewas essential for the imprinting and later olfactory-based kinrecognition process. Without visual exposure to other kin, there wasno kin preference and recognition later in life (Fig. 1D). Interestingly,visual cues of same-aged non-kin did not evoke imprinting (Fig. 1G).Again, we identified the time period for visual imprinting, whichturned out to happen at 5 dpf (Hinz et al., 2013a). Both olfactory andvisual cues were essential and larvae did not express any kinrecognition when the cues were given at any other time than 5 dpf(visual cue) and 6 dpf (olfactory cue) or when originating from non-kin. These findings indicate that a genetic predisposition exists thatresults in imprinting when larvae experience the right cues.

Our results about the necessity of visual kin cues were intriguing.How can a larva know what its siblings look like when it has neverseen any larvae before?What makes them know that the other larvae

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outside its own beaker are relatives? To understand thisphenomenon, we raised single fertilized eggs/larvae in glassbeakers surrounded by mirrors – we added holding water of kin at6 dpf and obtained strong kin preference (Fig. 1H). Without addingkin odour, we did not observe kin recognition (Fig. 1I). Weconcluded that self-matching on chemical cues is not possible, butself-matching on visual cues is possible.We wondered whether fish did not imprint on their own body

odour because the concentration of urine from a single larva is toolow to trigger imprinting. To test this possibility, we kept two fullsiblings in twice as much water as a single larva, to obtain thesame concentration, and found that concentration had no effect:imprinting was observed when two siblings were kept together butnot when a larva was kept individually, although a view ofadditional siblings outside the beaker was possible in bothconditions.To understand the visual part of imprinting, we took photos from

the lateral side of larvae and compared the pigment pattern withinand between sibling groups; this showed that related larvae share asimilar pigmentation pattern (Hinz et al., 2012). The highestsimilarity among related larvae was found in the pigmentationpattern of the eye, indicating that the process is based on geneticdisposition rather than direct comparison of own-body pigmentationwith that of others. Larvae in a natural environment cannot see their

own eye pigmentation in contrast to larvae in the above-mentionedmirror experiments.

Advantage of kin groupsWhat is the selective force for olfactory imprinting and resulting kinrecognition in zebrafish larvae? Larvae raised in groups of full-siblings showed an increased growth rate of 33% compared withlarvae raised with non-kin (Gerlach et al., 2007b); however, wecould not observe any differences in aggression or foraging.Therefore, association with kin and faster growth rates might lead toenhanced fitness by earlier reproduction. In Atlantic salmon andrainbow trout, kin group members had more foraging opportunitiesand experienced significantly greater weight gain than non-kingroup members (Brown and Brown, 1996). In three-spinedsticklebacks (Gasterosteus aculeatus), Frommen and Bakker(2004) suggested that shoaling with kin reduces competition,allows for more stable dominance hierarchies and increases fitnessof relatives.

MHC class II peptide ligands as chemical signals evoking olfactoryimprinting and kin recognitionIn vertebrates, much effort has been made to reveal which cues areused to identify kin. The kin signal must fulfil two attributes: it mustbe sufficiently unique to avoid mistakes and it has to be stable in

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Fig. 1. Summary of behavioural olfactory kin preference tests. A–O are modified from Hinz et al., 2012, 2013a,b. The diagrams on the left illustrate theraising conditions, which are described in the next two columns (vis., visual cues; olf., olfactory cues). ‘Test conditions’ describe chemical cues that larvaeexperienced during the test. ‘Olfactory preference’ indicates whether larvae expressed a statistically significant preference for kin or no preference; see ‘Olfactoryimprinting on conspecific and kin cues in zebrafish’ for details. MHC II, major histocompatibility complex (class II).

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order to be recognized over space and time. In the aquaticenvironment, these are non-volatile olfactory cues. The expressionof genes of the major histocompatibility complex (MHC) providesindividuals with a unique body odour that is more similar amongindividuals with increasing genetic relatedness (Apanius et al.,1997; Boehm and Zufall, 2006; Penn, 2002; Singh, 2001).Imprinting on MHC and other relatedness cues released via urine,mucus and gills plays a major role in kin recognition in fish andother aquatic species (for review, see Gerlach and Hinz, 2012).The olfactory preference of individual zebrafish larvae appears to

be defined at least in part by their MHC class II genotype.We used acombination of artificial MHC peptides to study their potential rolein imprinting (Hinz et al., 2013b). First, we searched for siblinggroups, which developed a preference for a specific set of fiverandomly chosen synthetic MHC peptides when they had beenexposed to these peptides during the critical period for imprinting.We found one zebrafish line (OL1, one pair and their offspring) thatshowed olfactory preference after exposure to these peptides at 6 dpf(Hinz et al., 2013b) (Fig. 1J). This exposure resulted not only in asubsequent preference for the MHC peptide mix but also in apreference for kin water (Fig. 1K), suggesting that the chosen MHCpeptides are identical or similar to components of the kin-specificodour cues that are relevant for imprinting in this line (Fig. 1).We further examined whether zebrafish can imprint on olfactory

cues of other unrelated zebrafish that bear the same MHC genotype.The similarity of MHC class II but not MHC class I alleles betweentwo zebrafish larvae corresponded to imprinting. Larvae did notdiscriminate between water derived from fish with identical MHCclass II alleles even though they differed in genetic relatedness(Hinz et al., 2013b) (Fig. 1L–O). Hence, the MHC class II genotypeappears to code for important olfactory cues for kin recognition andimprinting in zebrafish.Interestingly, MHC class II allele sharing led to similarity not

only in olfactory cues but also in pigmentation pattern. The degreeof allele sharing of MHC class II genes defined body, rump, tail andiris pigmentation (Hinz et al., 2012).We concluded that the MHC class II genotype could represent the

underlying mechanism carrying the information about kin and non-kin and, thus, be the basis of the genetic predisposition (Hinz et al.,2013b).

Neuronal basis of olfactory imprintingOlfactory system and its signal transduction in fishUnlike that in mammals, the olfactory system of the zebrafish as ateleost consists of only one main olfactory organ (Hansen et al.,2004; Hansen and Zeiske, 1993, 1998). Olfactory chemoreceptionis dependent on the binding of an odorant to its correspondingreceptors, located on microvilli or cilia of olfactory sensory neurons(OSNs), with subsequent signal transmission into the centralnervous system. Olfactory receptors (ORs) are located on thedendrites of OSNs embedded in the olfactory epithelium (OE) (seeFig. 2). Two major populations of OSNs are present in fish –microvillous OSNs (mOSNs) and ciliated OSNs (cOSNs) –resembling OSNs present in tetrapod vomeronasal and mainolfactory systems (Eisthen, 1997; Korsching, 2016). Zebrafishfeature two more OSN types: crypt cells (Ahuja et al., 2013; Hansenand Finger, 2000; Kress et al., 2015) and kappe cells (Ahuja et al.,2014) – both are named after their peculiar morphology and are sofar only known to be present in fish (see Fig. 2). Odorantinformation is transduced into electrical signals inside the OSNs.OSNs in the nose express one olfactory receptor out of a largerepertoire and project directly to the first olfactory processing centre

in the brain, the olfactory bulb (OB), where many of them convergeinto fewer glomeruli (primary olfactory pathway) (Yoshihara,2009). Bulbar projections further transmit odorant information intodifferent areas of the telencephalon and diencephalon (secondaryolfactory pathway) (Yoshihara, 2009). For example, in carp,mOSNs are mainly tuned towards food-related odours, whereascOSNs play a role in mediating the alarm reaction, and crypt cellsare suggested to be involved in reproduction (Hamdani et al., 2001,2008; Hamdani and Døving, 2002, 2006). Electrophysiological datafrom Hansen et al. (2003) in channel catfish suggest that all OSNtypes respond to amino acids, with cOSNs additionally respondingto bile salts, whereas mOSNs also respond to nucleotides. Ingoldfish, mOSNs are assumed to respond preferentially to aminoacids (Speca et al., 1999). In transgenic zebrafish, blocking ofsynaptic transmission in distinct populations of mOSNs abolishedattractive behavioural responses to amino acids (Koide et al., 2009),which is in line with the findings of other studies (Lipschitz andMichel, 2002). Amino acids and nucleotides are typically indicativeof food whereas bile salts are considered as social odorants as bilesalt profiles within a teleost family and order show high similarity(Hagey et al., 2010). However, a general conclusion on teleost OSNtuning is hard to make as the response profiles of OSNs differ, as dobulbar projection patterns across species (Bazáes et al., 2013).

Experience of olfactory cues can induce changes in the olfactoryneuronal systemThe olfactory system of teleosts enables the recognition anddiscrimination of a vast number of environmental and social odours.In the aquatic realm, olfactory-based behavioural context includesmigration (Yamamoto et al., 2010), association with kin (Hinz et al.,2013b; Olsen et al., 2002), feeding (Miklavc and Valentincic, 2012),predator avoidance (Idler et al., 1956), response to alarm signals(Schreckstoff) (Speedie and Gerlai, 2008) and social interactions suchas mating and reproduction (Veyrac et al., 2011). The olfactory systemof fish detects and discriminates between structurally different classesof odorants: mainly charged molecules including amino acids, bileacids, nucleotides, steroids and prostaglandins, but also unchargedvolatile molecules (Korsching, 2016). Responses to amino acids, bileacids and nucleotides (Carr, 1988) emerge early in development,shortly before hatching (Li et al., 2005). In zebrafish and other teleosts,defined prostaglandins and steroids serve as pheromonal signals priorto mating (Kobayashi et al., 2002; Moore andWaring, 1996; Sorensenet al., 1988; Stacey et al., 1989; Yabuki et al., 2016), and substancesreleased from the skin evoke an alarm response (Jesuthasan andMathuru, 2008). Most of the behavioural responses to these stimuliarise late in development, but the emergence of physiological andneurobiological responses to these stimuli has not been studied indetail. However, Braubach et al. (2013) provided exciting evidence ofan activity-dependent developmental mechanism in the zebrafisholfactory system, which might be important to understand olfactoryimprinting. They suggested that imprinting could trigger neuronalchanges in the OB and perhaps in the expression of specific olfactoryreceptor genes dependent on contact with olfactory cues at a specifictime during development. Ochs et al. (2017) described themorphology of glomerular patterning in wild Chinook salmonduring a temporal window previously shown to be significant forearly olfactory imprinting.

Sockeye salmon (Oncorhynchus nerka) that successfully imprintedon L-arginin, as evidenced by adult behaviour, demonstrated increasedexpression (relative to arginine-naive fish) of the putative argininereceptor mRNA in theOE during key life stages (Dittman et al., 2010).In brains of hatchery-reared juvenile chum salmon (Oncorhynchus

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Fig. 2. Zebrafish odorant receptors and olfactory sensory neurons (OSNs). (A) Olfactory epithelial positions of zebrafish OSNs. Combinatorial analysis ofcalcium-binding protein (CBP) expression reveals at least eight subpopulations of OSNs. Bold font indicates the largest of three subpopulations of ciliated OSNs(parvalbumin/calbindin/calretinin, PV/CB/CR; red). Four subpopulations of microvillous OSNs (mOSNs, dark blue) express PV. CBP S100 is specificallypresent in all crypt cells (green) and aminor subpopulation of mOSNs, which also express PV (shown again on the right of crypt cells in light blue). Kappe neurons(purple) probably express PV and CB and probably correspond to the PV/CB-mOSN population. (B) Zebrafish olfactory sensory cell types defined bymorphologyand CBPs. Schematic drawing of a cross-section through a larval zebrafish olfactory epithelium and olfactory bulb of one body side. The mediodorsalglomerular field (mdG) is indicated by six glomeruli (1–6). A hypothetical representation of the crypt cells (green) projecting to mdG2 is shown. Additional axonalinput to this glomerulus comes fromS100/PV-expressingmicrovillous OSNs (light blue). A second glomerulus (out of the 6) within themediodorsal domain, mdG5(5), receives input from PV/CB-expressing OSNs, probably representing kappe neurons. Also shown are the ventroposterior glomerulus (vpG) and theventrolateral (VLF) and ventromedial (VLM) glomerular fields subserved by mOSN and cOSN input, respectively. See text and Kress et al. (2015) for moredetails. (C) Comparison of zebrafish olfactory receptors and OSNs with those in mouse and human (for sources of data, see ‘Olfactory system and its signaltransduction in fish’).

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keta), thyrotropin-releasing hormone gene expression increasedimmediately after release from a hatchery into the natal stream, andthe expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor (NMDAR) increased during downstream migration(Ueda et al., 2016). The NMDAR is a glutamate receptor channelsubtype andmediates most of the fast-excitatory synaptic transmissionin the central nervous system. It plays important roles in memoryformation and retrieval in fish (Gómez et al., 2006; Kinoshita et al.,2004). Gene expression of salmon gonadotropin-releasing hormone(sGnRH) and NR1 increased in the adult chum salmon brain duringhoming from the Bering Sea to the natal hatchery (Ueda et al., 2016).However, in contrast to salmonids, in zebrafish, we could not evokeany olfactory preference for a variety of amino acids after larvae wereexposed to different concentrations of these amino acids duringdevelopment (K.T. and G.G., unpublished data). However, zebrafishas a non-migrating fish might imprint on other cues.

Tuning of teleost OSNs and processing of kin odour in thezebrafish OETo investigate which type of OSN detects kin odour, we mappedneuronal activity following olfactory stimulation visualized bypERK upregulation in OSNs in the OE of 9 dpf zebrafish larvae(Biechl et al., 2016). pERK is widely used to mark neuronal activityin mammals; for example, in the olfactory system following odourstimulation in mice (Mirich et al., 2004). Upon phosphorylation,pERK is translocated into the nucleus of the activated cell tomodulate the expression of transcription factors, which in turnregulate the expression of genes involved in neuronal and synapticplasticity underlying learning and memory. pERK is preferable toother markers, such as immediate early genes (e.g. c-fos or egr1),because of its more rapid activation and its cellular distribution(soma and cell protrusions) (Gao and Ji, 2009; Randlett et al., 2015).Olfactory stimulation with different odours (food, conspecific

odour or E3-medium) showed a differential pattern of activatedOSNs within the larval OE (Biechl et al., 2016). High numbers ofcOSNs – and a smaller number of mOSNs – were activated inresponse to food odour exposure, whereas mOSNs almostexclusively showed pERK upregulation following exposure to aconspecific odour (Biechl et al., 2016). Interestingly, crypt cellsshowed neuronal activation in response to neither food norconspecific (non-kin) odour. These response patterns weregenerally consistent with data from other studies, despite greatinterspecific variability regarding the tuning of OSNs withinteleosts (Bazáes et al., 2013).By rearing larvae isolated in small glass beakers in a larger tank,

we either prevented olfactory imprinting on day 6 or allowed thelarvae to imprint on their kin by adding kin odour containing waterto the beakers (Biechl et al., 2016). The rearing conditions for alllarvae were otherwise identical (e.g. changing water, feeding andisolation), precluding other influences on the results. Importantly,despite raising larvae in glass beakers, visual imprinting at 5 dpf waspossible because isolated larvae could see their siblings swimmingoutside their own beakers in the larger tank (see also Hinz et al.,2013a). Additionally, in the subsequent histological assay for pERKwe used the calcium-binding protein (CBP) S100 to specificallyidentify crypt cells. In the crucian carp (Carassius carassius), cryptcells project into the ventral OB from where projection neuronsterminate in the lateral part of the medial olfactory tract, which isknown to mediate reproductive behaviours (Weltzien et al., 2003).Furthermore, in sexually mature carp, crypt cells were shown to varyin their density as well as location within the OE depending on theseason (Hamdani and Døving, 2006; Hamdani et al., 2008). Carp

exhibited only a few such cells during winter whereas in the summerspawning season, carp crypt cells were clearly detectable andpositioned at the surface of the OE, suggesting that they wereinvolved in carp reproductive behaviour (Hamdani et al., 2008).Sandulescu et al. (2011) demonstrated an interesting time course ofzebrafish crypt cell quantity during OE development. Upon firstappearance in the zebrafish OE at 4 dpf, the number of crypt cellsincreased steadily until reaching a peak at 7 dpf. Comparing thisgrowth of crypt cells with the sensitive phase of olfactory imprinting(day 6), it appears as though crypt cell numbers might adapt to theupcoming imprinting event.

Based on these studies, we exposed imprinted and non-imprintedzebrafish larvae at 9 dpf to kin odour; only crypt cells of imprintedlarvae showed activation in response to kin odour (Fig. 3C; Biechlet al., 2016). Importantly, this was not due to an absence of this celltype, as crypt cell numbers did not differ between non-imprinted andimprinted larvae (Biechl et al., 2016) (Fig. 3A). Furthermore,microarray data provided no evidence for a down-regulation ofORA4 receptor expression in crypt cells in non-imprinted comparedwith imprinted zebrafish larvae (K.T. and G.G., unpublished data).Thus, the critical cue, which is obviously contained in kin odour,apparently changed the responsiveness of crypt cells in an unknownmanner during the imprinting process. Crypt cells express only a singleV1R-like receptor, encoded by the ora4 gene (Oka et al., 2012). Theligand that binds to ORA4 is still unknown; however, based on ourdata, the ligand of ORA4 is contained in kin odour but it is unlikelythat ORA4 is the specific receptor for MHC peptides in zebrafish(Behrens et al., 2014; Boschat et al., 2002; Isogai et al., 2011).

In addition to crypt cells, a small subpopulation of (S100-negative)mOSNs was shown to respond to kin odour (Fig. 3D). Consideringthat MHC peptides are a component of kin odour, these mOSNsmight express a V2R receptor and bind to such peptides. Regardless,kin odour is composed of a mixture of numerous odorants that signal– besides genetic relatedness – additional social information such assex and physiological status, and possibly kin recognition is mediatedby more than one chemical compound. Thus, a receptor code,generated by the interaction of multiple receptors and activated bymore than one ligand, might signal information about relatedness inlarval zebrafish. In sum, our studies support the hypothesis that cryptcells as well as a small subpopulation of mOSNs are involved indetecting a kin odour-related signal.

Potential neuronal changes due to olfactory imprintingNevitt et al. (1994) hypothesized a model for olfactory imprinting insalmon. They suggested that during sensitive imprinting periods,thyroid hormones stimulate the proliferation of non-specific olfactorysensory neurons. Subsequently, the receptors that are most responsiveto the odorants survive and the non-responsive receptors die. Thismodel was based on previous studies that demonstrated odorant-specific sensitization of the peripheral olfactory system in cohosalmon (Oncorhynchus kisutch) (Dittman et al., 1997). Their resultssuggested that exposure of coho salmon to an odorant for 10 daysduring smolting could result in significant changes in the peripheralsensitivity to that odorant in the spawning adult coho salmon(Dittman et al., 2010, 1997).

We tested whether the expression of odorant receptors and othergenes in zebrafish larvae changed during the sensitive phase ofolfactory imprinting through a genome-wide transcriptional analysisusingmicroarray chip that included gene-level probe sets for 129 OR,82 TAAR, 46 OlfC and 6 ORA odorant receptor genes, covering allknown ora genes, but missing probe sets for some odorant receptorgenes from the TAAR, OR and OlfC group (K.T. and G.G.,

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unpublished results). Microarray analysis showed no differences ingene expression for 263 odorant receptor genes in imprinted versusnon-imprinted larvae (K.T. and G.G., unpublished results); however,we might have missed specific odorant receptors especially someTAARs, which are suggested to be associated with the detection ofsocial cues in mice (Ziegler et al., 2002).Zebrafish larvae exposed to the artificial odorant β-phenethyl

alcohol (βPEA) showed an increase in expression of the transcriptionfactor Otx2 inside the OE (Calfún et al., 2016; Harden et al., 2006).The authors suggested that βPEA triggers olfactory imprinting injuvenile zebrafish and further showed that three OR genes (OR111-1,OR115-5, OR125-1) are affected by Otx2 expression.Our qPCR analysis in 5, 6 and 7 dpf zebrafish larvae (before,

during and after olfactory imprinting) revealed no differences inOtx2 expression; the same finding was obtained for imprinted andnon-imprinted larvae (K.T., G.S and A.S., unpublished results).In situ hybridization experiments confirmed our findings: nodifferences in Otx2 expression pattern were observed in 5, 6 and7 dpf imprinted and non-imprinted larvae.

Spatial distribution of odour responses in the OBTo explore how olfactory cues relevant for imprinting are representedin the zebrafish brain, we measured odour-evoked activity in the OBand adjacent areas by multiphoton calcium imaging (Hinz et al.,2013b). Patterns of input activity across the array of glomeruli areprocessed by neuronal circuits in the OB that are composed ofprincipal neurons, the mitral cells and multiple types of interneurons.General odorants such as amino acids, bile acids and nucleotidesstimulate multiple glomeruli, and stimuli of the same class often evoke

responses in overlapping, yet stimulus-specific, subpopulations of OBneurons (Friedrich, 2013).

The spatial distribution of odour responses is usually broad butnot entirely random because responses to odours of the same classare biased towards broad sub-regions. For example, responses toamino acids and bile acids are found predominantly in theventrolateral and the dorsomedial OB, respectively (Doving et al.,1980; Friedrich and Korsching, 1997, 1998a; Yaksi et al., 2009).This coarse functional topography of odour responses is establishedat early stages of development and maintained throughoutadulthood (Li et al., 2005). Pheromones, in contrast, have beenreported to activate few glomeruli with high specificity (Friedrichand Korsching, 1998b; Yabuki et al., 2016). Moreover, pheromonesand general odorants activate different brain areas downstream ofthe OB (Yabuki et al., 2016), consistent with the hypothesis thatpheromonal signals are detected by specific receptors and controldistinct behavioural or endocrine responses.

Neuronal representation of olfactory kin cues in the OBIn mammals, MHC peptides convey information about individualityand stimulate sensory neurons of the main and accessory olfactorysystems (Leinders-Zufall et al., 2004; Spehr et al., 2006). Individualsensory neurons respond tomultiple peptides, indicating that stimulusidentity is encoded by a combination of activity patterns acrossmultiple odorant receptors and glomerular channels. However, thespatial distribution of responses to MHC peptides in the OB has notbeen analysed in detail. To address this, we mapped odour responsesto a mixture of the five MHC peptides that evoked olfactoryimprinting and kin recognition in a specific kin group (Hinz et al.,

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100Fig. 3. Differential activation of cryptcells with kin odour – effects ofolfactory imprinting. (A) Total cellquantity of S100+ mOSNs and crypt cells.Imprinting has no effect on total cellnumber. (B) Number of S100+/pERK+mOSNs shown as a percentage of allS100+ mOSNs per larva. Significantlymore S100+ mOSNs are activated inimprinted larvae versus non-imprintedcontrol larvae exposed to kin odour. (C) Asignificantly higher number of crypt cells isactivated after kin odour stimulation inimprinted compared with non-imprintedlarvae and compared with imprintedcontrol larvae stimulation. No difference inactivation was found within non-imprintedlarvae. (D) The total number of pERK+, butS100−, ciliated OSNs (cOSNs), mOSNsand crypt cells. Cell activation was similarfor all treatments in cOSNs. A significantlyhigher number of mOSNs was found inimprinted larvae stimulated with kincompared with control stimulation. NoS100− crypt cells were observed.Statistical significance: **P<0.01,***P<0.001. Modified from Biechl et al.(2016).

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2013b) in the OB by multiphoton calcium imaging in transgenic fishexpressing the genetically encoded calcium indicator GCaMP2 underthe control of the pan-neuronal elavl3 promoter (Ahrens et al., 2012)(Fig. 4). Because zebrafish larvae are small and transparent, activitycould be imaged in multiple focal planes, separated by 10 µm, thatcovered the entire OB and parts of the telencephalon and habenula invivo. Results from multiple fish were combined by registration ofimage data to a common reference brain (Fig. 4D). We found thatresponses to MHC peptides were distributed and not confined to aspecific location, although responses appeared to be particularlydense in a sub-volume of the ventrolateral, amino acid-responsivepart of the OB (Fig. 4) (Hinz et al., 2013b).

Unlike responses to amino acid odours, responses to MHCpeptides did not substantially broaden with increasing stimulusconcentration (Hinz et al., 2013b), consistent with observations inmammalian sensory neurons (Leinders-Zufall et al., 2004). The bestresponse was observed at very low concentrations of MHC peptidemix (1.25×10−12 mol l−1) (Hinz et al., 2013b). Activity patternsevoked by MHC peptides showed significant overlap with activityevoked by the odour of conspecifics (Fig. 4), but less overlap withactivity evoked by food extract (Hinz et al., 2013b), even thoughfood extract is a strong and complex odour. These observationssupport the hypothesis that MHC peptides are released into thewater and detected by the olfactory system of zebrafish. Moreover,

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Fig. 4. Spatial distribution of responses to MHC peptides and fish odour. (A,B) Expression of GCaMP2 (greyscale) and odour-evoked relative change influorescence (colour; thresholded) in four optical sections of transgenic zebrafish. A depth of z=0 corresponds to the dorsal pole of the olfactory bulb; negativevalues indicate more ventral locations. Right: maximum projection of responses in 10 optical planes spaced at 10 µm. Stimuli were water conditioned byconspecifics (fish odour; A) and a mix of five MHC peptides (B). All fish were raised in the presence of siblings. In trial- and time-averaged response maps, pixelswere thresholded at 2.5 s.d. and contiguous areas of less than 20 pixels (∼2.8×2.8 µm2) were deleted. (C) Intersection of activity patterns evoked by fishodour and theMHC peptide mix. In each optical section, response intensities were summedwhen relative changes in fluorescence exceeded 2.5 s.d. in responseto both stimuli. Right: maximum projection of responses in 10 optical sections spaced at 10 µm. (D) Alignment of images from multiple individuals. The fourlandmarks L1–L4 were aligned by translation, scaling and rotation of image data. OB, olfactory bulb; Tel, telencephalon; Hb, habenula; A, anterior; P, posterior;R, right; L, left. (E) Depth histogram of optical planes imaged in six fish (10 µm spacing). (F–H) Thresholded and averaged responses to fish odour (F) orto MHC peptide mix (G); (H) intersection. Maximum projections through 10–11 optical planes in each of six fish were aligned and combined by taking maximumvalues. Optical sections covered the entire olfactory bulb but only parts of the telencephalon and habenula.

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unlike pheromones, MHC peptides do not appear to be detected by asmall number of narrowly tuned sensory channels. Rather, MHCpeptides appear to be represented in a combinatorial fashion byactivity patterns across neuronal populations, consistent withobservations in the peripheral olfactory system of mammals(Leinders-Zufall et al., 2004; Spehr et al., 2006).As the observed higher neuronal activity in specific OSNs (crypt

cells and mOSNs) in imprinted larvae is not explained by anincreased number of these OSNs or a change in odorant receptor geneexpression, olfactory imprinting might be explained by a change insensitivity of the odorant receptor itself. A ligand–MHC protein–odorant receptor interaction might lead to a stronger neuronalactivation compared with that in larvae that are exposed to peptideligands of non-kin odour. Odorant-binding proteins can enhance thesolubility of hydrophobic odours and facilitate the transport of odoursto receptor sites (Pelosi, 1994). Even if this does not represent amodulation of receptor affinity, it still enhances the possibility of aligand–receptor interaction and thereby might represent a station ofneuronal activity modulation in general.Our approach to study processes that underlie olfactory

imprinting and kin recognition in a non-migratory but much moretractable fish should shed light on the mechanistic underpinnings ofimprinting in parental populations in migratory fishes. But olfactoryimprinting on kin versus environmental cues seems to differ, asshown by differences in olfactory receptor gene expression.

AcknowledgementsSome parts of the results and discussion are taken from the PhD thesis of DanielaBiechl (Ludwig Maximilian University, Munich, 2018). We thank Sigrun Korsching forfruitful discussions. We also thank B. Grothe for additional support. We thank PascalFieth for bioinformatics support for the microarray gene expression analysis. We aregrateful to R. Friedrich and Mario Wullimann for their contributions to the discussion.

Competing interestsThe authors declare no competing or financial interests.

FundingFunding was provided by the Deutsche Forschungsgemeinschaft DFG (SPP 1392IntegrativeAnalysis ofOlfaction;GE842/5-1, 5-2, FR1667/2-1, 2-2andWU211/2-1, 2-2).

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