Expression of olfactory receptors in different life stages and life histories of wild Atlantic salmon (Salmo salar)

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory-related genes in the olfactory rosettes of different life stages in two anadromous and one non-anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non-anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory-related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.     

Differential expression of olfactory genes in Atlantic salmon (Salmo salar) during the parr–smolt transformation

The anadromous salmon life cycle includes two migratory events, downstream smolt migration and adult homing migration, during which they must navigate with high precision. During homing migration, olfactory cues are used for navigation in coastal and freshwater areas, and studies have suggested that the parr – smolt transformation has a sensitive period for imprinting. Accordingly, we hypothesized that there would be significant changes in gene expression in the olfactory epithelium specifically related to smoltification and sampled olfactory rosettes from hatchery‐reared upper growth modal juvenile Atlantic salmon at 3‐week intervals from January to June, using lower growth modal nonsmolting siblings as controls. A suite of olfactory receptors and receptor‐specific proteins involved in functional aspects of olfaction and peripheral odor memorization was analyzed by qPCR. Gene expression in juveniles was compared with mature adult salmon of the same genetic strain caught in the river Gudenaa. All mRNAs displayed significant variation over time in both modal groups. Furthermore, five receptor genes (olfc13.1, olfc15.1, sorb, ora2, and asor1) and four olfactory‐specific genes (soig, ependymin, gst, and omp2) were differentially regulated between modal groups, suggesting altered olfactory function during smoltification. Several genes were differentially regulated in mature salmon compared with juveniles, suggesting that homing and odor recollection involve a different set of genes than during imprinting. Thyroid hormone receptors thrα and thrβ mRNAs were elevated during smolting, suggesting increased sensitivity to thyroid hormones. Treatment of presmolts with triiodothyronine in vivo and ex vivo had, however, only subtle effects on the investigated olfactory targets, questioning the hypothesis that thyroid hormones directly regulate gene expression in the olfactory epithelium.     

Genomic organization and characterization of two vomeronasal 1 receptor-like genes (ora1 and ora2) in Atlantic salmon Salmo salar

Olfactory receptors are encoded by three large multigene superfamilies (OR, V1R and V2R) in mammals. Fish do not possess a vomeronasal system; therefore, it has been proposed that their V1R-like genes be classified as olfactory receptors related to class A G protein-coupled receptors (ora). Unlike mammalian genomes, which contain more than a hundred V1R genes, the five species of teleost fish that have been investigated to date appear to have six ora genes (ora1-6) except for pufferfish that have lost ora1. The common ancestor of salmonid fishes is purported to have undergone a whole genome duplication. As salmonids have a life history that requires the use of olfactory cues to navigate back to their natal habitats to spawn, we set out to determine if ora1 or ora2 is duplicated in a representative species, Atlantic salmon (Salmo salar). We used an oligonucleotide probe designed from a conserved sequence of several teleost ora2 genes to screen an Atlantic salmon BAC library (CHORI-214). Hybridization-positive BACs belonged to a single fingerprint contig of the Atlantic salmon physical map. All were also positive for ora2 by PCR. One of these BACs was chosen for further study, and shotgun sequencing of this BAC identified two V1R-like genes, ora1 and ora2, that are in a head-to-head conformation as is seen in some other teleosts. The gene products, ora1 and ora2, are highly conserved among teleosts. We only found evidence for a single ora1-2 locus in the Atlantic salmon genome, which was mapped to linkage group 6. Fluorescent in situ hybridization (FISH) analysis placed ora1-2 on chromosome 12. Conserved synteny was found surrounding the ora1 and ora2 genes in Atlantic salmon, medaka and three-spined stickleback, but not zebrafish.     

Crypt neurons express a single V1R-related ora gene

Both ciliated and microvillous olfactory sensory neuron populations express large families of olfactory receptor genes. However, individual neurons generally express only a single receptor gene according to the "one neuron-one receptor" rule. We report here that crypt neurons, the third type of olfactory neurons in fish species, use an even more restricted mode of expression. We recently identified a novel olfactory receptor family of 6 highly conserved G protein-coupled receptors, the v1r-like ora genes. We show now that a single member of this family, ora4 is expressed in nearly all crypt neurons, whereas the other 5 ora genes are not found in this cell type. Consistent with these findings, ora4 is never coexpressed with any of the remaining 5 ora genes. Furthermore, several lines of evidence indicate the absence of any other olfactory receptor families in crypt neurons. These results suggest that the vast majority of the crypt neuron population may select one and the same olfactory receptor gene, a "one cell type-one receptor" mode of expression. Such an expression pattern is familiar in the visual system, with rhodopsin as the sole light receptor of rod photoreceptor cells, but unexpected in the sense of smell.     

Molecular biological research on olfactory chemoreception in fishes

This review describes recent molecular biological research on olfactory chemoreception in fishes. The recent rapid development of molecular biological techniques has provided new valuable information on the main and vomeronasal olfactory receptor (OR) genes, the axonal projection from ciliated, microvillous and crypt-olfactory receptor cells to the olfactory bulb, properties of odorant substances and olfactory imprinting and homing in salmon. Many important questions, however, remain unanswered on functional differences among OR genes, on ligand binding to each OR and on the molecular biological mechanisms underlying olfactory imprinting and homing in salmon. Olfactory chemoreception is believed to be the oldest sensory cue for both animal survival and adaptation to various different environments. Further intensive molecular biological research on olfactory memory formation and remembrance should be carried out to clarify the fundamental process of olfactory chemoreception in fishes.     

L-alanine binding sites and Na+, K(+)-ATPase in cilia and other membrane fractions from olfactory rosettes of Atlantic salmon.

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.     

Olfactory gene expression in migrating adult sockeye salmon Oncorhynchus nerka

Expression of 12 olfactory genes was analysed in adult sockeye salmon Oncorhynchus nerka nearing spawning grounds and O. nerka that had strayed from their natal migration route. Variation was found in six of these genes, all of which were olfc olfactory receptors and had lower expression levels in salmon nearing spawning grounds. The results may reflect decreased sensitivity to natal water olfactory cues as these fish are no longer seeking the correct migratory route. The expression of olfactory genes during the olfactory‐mediated spawning migration of Pacific salmon Oncorhynchus spp. is largely unexplored and these findings demonstrate a link between migratory behaviours and olfactory plasticity that provides a basis for future molecular research on salmon homing.     

Molecular characterisation of a putative Atlantic salmon (Salmo salar) odorant receptor

The olfactory system of fish is extremely important as it is able to recognise and distinguish a vast array of odorous molecules that are involved in behaviours paramount to survival. This is achieved by the activation of a diverse multigene family of G-protein coupled receptors through odorous ligand binding. Using molecular techniques, the nucleotide sequence of the cDNA coding for an Atlantic salmon (Salmo salar) odorant receptor (ASOR1) has been determined. The full-length cDNA (1260 nt) encodes a protein of 320 amino acid residues, including one potential N-linked glycosylation site, within the short extracellular amino terminal of the receptor. Hydrophobicity analysis revealed seven hydrophobic regions within the amino acid sequence, corresponding to possible positions of the transmembrane domains characteristic of the G-protein coupled receptor superfamily. Several conserved motifs unique to odorant receptors were also present. Through characterisation of this receptor, we hope to increase the understanding of the mechanisms underlying olfaction in salmonid species.     

Sequence conservation among orthologous vomeronasal type 1 receptor-like (ora) genes does not support the differential tuning hypothesis in Salmonidae

Salmon utilize olfactory cues to guide natal stream homing during spawning migrations. Both inorganic and biogenic chemicals have been proposed as odorants that might be used by salmon during homing. In this study, we used genomic DNA sequence data from nine salmonid species to compare nucleotide identities for orthologous main olfactory receptor (mOR) genes with nucleotide identities for orthologous vomeronasal type 1-like (ora) receptor genes. We found that orthologs for both classes of olfactory receptor genes (mORs and Oras) appear to be highly conserved among species. Our findings do not support the differential tuning hypothesis in Salmonidae, which predicts higher sequence conservation for mORs than ora. We did, however, find convincing evidence for site-specific positive selection acting on paralogous main olfactory receptor genes.     

Paradoxical role of C1561T glutamate carboxypeptidase II (GCPII) genetic polymorphism in altering disease susceptibility

Although olfaction could play a crucial role in underwater habitats by allowing fish to sense a variety of nonvolatile chemical signals, the importance of olfaction in species-rich cichlids is still controversial. In particular, examining whether cichlids rely on olfaction for reproduction is of primary interest to understand the mechanisms of speciation. In the present study, we explored the V1R (also known as ora) genes, which are believed to encode reproductive pheromone receptors in fish, in the genomes of Lake Victoria cichlids. By screening a bacterial artificial chromosome library, we identified all six intact V1R genes (V1R1 to V1R6) that have been reported in other teleost fish. Furthermore, RT-PCR and in situ hybridization analyses showed that all of the V1R genes were expressed in the olfactory epithelium, indicating that these receptors are functional in cichlids. These observations indicate that cichlids use V1R-mediated olfaction in some ways for their social behaviors.     

Divergent evolution among teleost V1r receptor genes

The survival of vertebrate species is dependent on the ability of individuals to adequately interact with each other, a function often mediated by the olfactory system. Diverse olfactory receptor repertoires are used by this system to recognize chemicals. Among these receptors, the V1rs, encoded by a very large gene family in most mammals, are able to detect pheromones. Teleosts, which also express V1r receptors, possess a very limited V1r repertoire. Here, taking advantage of the possibility to unequivocally identify V1r orthologs in teleosts, we analyzed the olfactory expression and evolutionary constraints of a pair of clustered fish V1r receptor genes, V1r1 and V1r2. Orthologs of the two genes were found in zebrafish, medaka, and threespine stickleback, but a single representative was observed in tetraodontidae species. Analysis of V1r1 and V1r2 sequences from 12 different euteleost species indicate different evolutionary rates between the two paralogous genes, leading to a highly conserved V1r2 gene and a V1r1 gene under more relaxed selective constraint. Moreover, positively-selected sites were detected in specific branches of the V1r1 clade. Our results suggest a conserved agonist specificity of the V1R2 receptor between euteleost species, its loss in the tetraodontidae lineage, and the acquisition of different chemosensory characteristics for the V1R1 receptor.     

Semaphorins and their receptors in olfactory axon guidance.

The mammalian olfactory system is capable of discriminating among a large variety of odor molecules and is therefore essential for the identification of food, enemies and mating partners. The assembly and maintenance of olfactory connectivity have been shown to depend on the combinatorial actions of a variety of molecular signals, including extracellular matrix, cell adhesion and odorant receptor molecules. Recent studies have identified semaphorins and their receptors as putative molecular cues involved in olfactory pathfinding, plasticity and regeneration. The semaphorins comprise a large family of secreted and transmembrane axon guidance proteins, being either repulsive or attractive in nature. Neuropilins were shown to serve as receptors for secreted class 3 semaphorins, whereas members of the plexin family are receptors for class 1 and V (viral) semaphorins. The present review will discuss a role for semaphorins and their receptors in the establishment and maintenance of olfactory connectivity.     

Olfactory receptors in aquatic and terrestrial vertebrates

In species representing different levels of vertebrate evolution, olfactory receptor genes have been identified by molecular cloning techniques. Comparing the deduced amino-acid sequences revealed that the olfactory receptor gene family of Rana esculenta resembles that of Xenopus laevis, indicating that amphibians in general may comprise two classes of olfactory receptors. Whereas teleost fish, including the goldfish Carassius auratus, possess only class I receptors, the `living fossil' Latimeria chalumnae is endowed with both receptor classes; interestingly, most of the class II genes turned out to be pseudogenes. Exploring receptor genes in aquatic mammals led to the discovery of a large array of only class II receptor genes in the dolphin Stenella Coeruleoalba; however, all of these genes were found to be non-functional pseudogenes. These results support the notion that class I receptors may be specialized for detecting water-soluble odorants and class II receptors for recognizing volatile odorants. Comparing the structural features of both receptor classes from various species revealed that they differ mainly in their extracellular loop 3, which may contribute to ligand specificity. Comparing the number and diversity of olfactory receptor genes in different species provides insight into the origin and the evolution of this unique gene family. Accepted: 29 July 1998     

Ontogenetic shifts in olfactory rosette morphology of the sockeye salmon,Oncorhynchus nerka

Sockeye salmon, Oncorhynchus nerka, are anadromous, semelparous fish that breed in freshwater—typically in streams, and juveniles in most populations feed in lakes for 1 or 2 years, then migrate to sea to feed for 2 or 3 additional years, before returning to their natal sites to spawn and die. This species undergoes important changes in behavior, habitat, and morphology through these multiple life history stages. However, the sensory systems that mediate these migratory patterns are not fully understood, and few studies have explored changes in sensory function and specialization throughout ontogeny. This study investigates changes in the olfactory rosette of sockeye salmon across four different life stages (fry, parr, smolt, and adult). Development of the olfactory rosette was assessed by comparing total rosette size (RS), lamellae number, and lamellae complexity from scanning electron microscopy images across life stages, as a proxy for olfactory capacity. Olfactory RS increased linearly with lamellae number and body size (p < .001). The complexity of the rosette, including the distribution of sensory and nonsensory epithelia and the appearance of secondary lamellar folding, varied between fry and adult life stages. These differences in epithelial structure may indicate variation in odor-processing capacity between juveniles imprinting on their natal stream and adults using those odor memories in the final stages of homing to natal breeding sites. These findings improve our understanding of the development of the olfactory system throughout life in this species, highlighting that ontogenetic shifts in behavior and habitat may coincide with shifts in nervous system development.     

Random expression of main and vomeronasal olfactory receptor genes in immature and mature olfactory epithelia of Fugu rubripes

Main olfactory receptor genes were isolated from a seawater fish, Fugu rubripes (pufferfish), and characterized. Two subfamilies of genes encoding seven transmembrane receptors were identified; one consists of five or more members, termed FOR1-1 to 5 of FOR1 subfamily, and the other appears to be a single copy gene, termed the FOR2 subfamily. FOR1 members show extremely high amino acid sequence similarities of about 95% to one another, and are distantly related to catfish-1 with the highest similarity of 37%. FOR2 shows 43% similarity to goldfish-A28. Phylogenically, both FOR members are categorized among pedigrees of the fish main olfactory receptor family outside the mammalian receptor family, although similarities between Fugu receptors and those of fresh-water fishes are lower than those among fresh-water fishes. In situ hybridization shows that both subfamilies of receptor genes are expressed randomly over the olfactory epithelium throughout all developmental stages, and no segregation of the signals was found. On the other hand, when three members of a vomeronasal olfactory receptor gene family, related to the Ca(2+)-sensing receptor, were used as probes, they were also randomly expressed over the same epithelium as the main olfactory receptors. This is in contrast to the expression profiles observed for zebrafish and goldfish, where the main or vomeronasal olfactory receptors are expressed in segregated patterns. It is thus suggested that the expression pattern of fish olfactory receptors varies depending on the species, although fish olfactory receptors are highly related to one another in their primary structures, and are phylogenically distinct from those of mammals.     

Migration of farmed adult Atlantic salmon with and without olfactory sense, released on the Norwegian coast

The dispersal and migration of farmed Atlantic salmon, Salmo salar , allowed to escape during the summer was studied. Three groups of 4–year–old fish of the River Imsa stock were released in coastal waters off south-western Norway: one group, with functional olfactory organs, was released at a fish farm 4 km away from the R. Imsa; two other groups, one with transected olfactory nerves and the other with functional olfactory organs, were released in the sea 90 km from the R. Imsa. To compare them with the migration pattern of reared, large smolts of the Imsa stock, a group of 3 + smolts was released in the R. Imsa.
Adults of salmon released as 3–year–old smolts homed with high precision to the R. Imsa. Four– year–olds released in the sea were recaptured in the fjord and in the coastal current, the majority north of the places of release. Immatures migrated to feeding areas in the North Atlantic. Matures seemed to enter rivers at random when ready to spawn. There was no difference in migration pattern between anosmics and controls. The olfactory sense was not mandatory for entering fresh water. The results indicate that the homing behaviour of Atlantic salmon is not a direct consequence of a single imprinting of the smolts, and that there is not a direct genetic link for return to a particular river. The present results support the sequential imprinting hypothesis proposed by Harden Jones (1968).     

Kin recognition in zebrafish: a 24-hour window for olfactory imprinting

Distinguishing kin from non-kin profoundly impacts the evolution of social behaviour. Individuals able to assess the genetic relatedness of conspecifics can preferentially allocate resources towards related individuals and avoid inbreeding. We have addressed the question of how animals acquire the ability to recognize kin by studying the development of olfactory kin preference in zebrafish (Danio rerio). Previously, we showed that zebrafish use an olfactory template to recognize even unfamiliar kin through phenotype matching. Here, we show for the first time that this phenotype matching is based on a learned olfactory imprinting process in which exposure to kin individuals on day 6 post fertilization (pf) is necessary and sufficient for imprinting. Larvae that were exposed to kin before or after but not on day 6 pf did not recognize kin. Larvae isolated from all contact with conspecifics did not imprint on their own chemical cues; therefore, we see no evidence for kin recognition through self-matching in this species. Surprisingly, exposure to non-kin odour during the sensitive phase of development did not result in imprinting on the odour cues of unrelated individuals, suggesting a genetic predisposition to kin odour. Urine-born peptides expressed by genes of the immune system (MHC) are important messengers carrying information about 'self' and 'other'. We suggest that phenotype matching is acquired through a time-sensitive learning process that, in zebrafish, includes a genetic predisposition potentially involving MHC genes expressed in the olfactory receptor neurons.