BACKGROUND
group living animals have to adjust the expression of social behavior to the nuances of daily social life and to transitions between life-history stages, and their ability to do so impacts on their darwinian fitness  <cit> .
this socially driven behavioral plasticity induces changes in brain neurogenomic states that underlie different behavioral repertoires  <cit> .
thus, reprogramming the transcriptome in response to the social environment allows an animal to switch between adaptive behavioral states  <cit> .
gene expression profiling enables the study of this dynamic relationship between genotype and behavior  <cit>  and to unveil the genetic networks behind complex behaviors.
in addition, the development of whole-genome sequencing, microarrays and other genomic resources for non-traditional model organisms, but with complex social repertoires, has provided relevant insights on how complex genotypes are translated to produce meaningful behaviors  <cit> .
in recent years, numerous studies have described the influence of social environment and of social interactions on transcriptional and neural activity  <cit> .
for example, caste differentiation  in the honey bee , a key feature in eusocial insects, is influenced not only by heritable traits but also by variations in the regulation of molecular pathways linked with several life-history traits, such as nutrition, metabolism, and reproduction  <cit> .
the activity of aggression-related genes in this species also seems to be under both inherited and environmental influences, varying with age, exposure to alarm-cues and depending on colony environment  <cit> .
the study of gene expression signatures of life history transitions has also been a focus in teleost fishes.
for example, life history traits of salmonids have also been addressed in a number of studies showing variation in brain expression profiles related with alternative reproductive and migratory tactics  <cit>  and their interaction with the rearing environment  <cit> .
all the results on the impact of the social environment on the transcriptome highlight new possibilities concerning how social stimuli, as well as more complex interactions between conspecifics, can influence and shape gene translation into producing appropriate behavioral responses, according to external and internal cues and also to the animals’ past experience.
most of the studies discussed above characterize fixed and irreversible behavioral phenotypes, which correspond to switches between “static” neurogenomic states.
but the interaction between the genome and the environment is also expected to be present in shorter time frames and to be reversible in order to accommodate labile and transient changes in behavioral states in order for flexible adaptive behavior to evolve  <cit> .
behaviorally, a single interaction may have consequences for the performance of the individuals and the outcome of future interactions , but its impact on the neurogenomic state of the individuals has been scarcely characterized.
animals integrate sensory information with internal physiology into context-appropriate behavior that ultimately promotes fitness.
yet how the brain integrates different sensory modalities in these social contexts remains unclear.
for example,  <cit>  presented males of the model cichlid astatotilapia burtoni with sensory information in three social contexts: intruder challenge, reproductive opportunity and a socially neutral situation.
the authors found that, compared to the neutral context, a visual stimulus was necessary and sufficient for an aggressive response, whereas chemical and visual stimuli presented alone were sufficient for an androgen response.
interestingly, the immediate-early gene c-fos, a neural activity marker, was induced in response to a visual challenge stimulus specifically in dopaminergic neurons of area vc , a putative striatal homologue, whereas presentation of a chemical stimulus alone did not induce c-fos expression in the intruder challenge context.
clearly, these results suggest that socially salient sensory cues are processed in a modality-dependent manner in the brain.
however, this study did not examine neuromolecular responses of forebrain regions associated with olfactory processing, such as the olfactory bulb or area dp .
the mozambique tilapia, oreochromis mossambicus, is an african cichlid fish that has become a model system in the study of neuroendocrine mechanisms underlying socially mediated behavioral changes .
the importance of chemical signaling of male social status has been described in this species , and the olfactory system, from sensory epithelium to bulbar and extrabulbar projections, has been well characterized  <cit> .
in nature, o. mossambicus males establish contiguous display territories, which females visit in order to obtain matings.
the repertoire of social behavior is highly complex and multimodal, including visual .
importantly, male tilapia store urine in their bladders which they use to signal social rank during agonistic interactions with other males or in the presence of pre-ovulatory females  <cit> .
furthermore, males are able to modulate their rate of urination depending on the social environment.
an increase of males’ urination rate is observed during agonistic encounters  <cit>  or in the presence of pre-ovulatory females  <cit> .
furthermore, both the volume of stored urine and its olfactory potency, as measured by electro-olfactogram  recordings, is higher in dom than in sub males  <cit> .
females do not store urine and have a higher frequency of urination  <cit> .
additionally, females have smaller kidneys, smaller urinary bladders and the urothelial thickness of the inner surface of the bladder is also smaller than in males  <cit> .
finally, the odor of pre-ovulatory females elicits higher amplitude eog responses in males than that of post-ovulatory females  <cit> .
depending on the social environment, tilapia males can exhibit two distinct behavioral phenotypes: dominants  and subordinate .
dom individuals adopt a typical velvet black coloration and establish breeding territories on the bottom, where they dig nests to which they attract females using courtship displays  <cit> .
sub males present a pale silver coloration and either move around among the breeding territories of dom males or shoal together with females, while they wait for their opportunity for social ascension.
sneaking fertilization attempts by sub males have also been reported  <cit> .
changes between these social phenotypes have been shown to activate a cascade of molecular processes and a variety of neuroendocrine pathways which include neuropeptides and steroid hormones  <cit> .
ovulated females visit male breeding arenas when ready to spawn and follow courting males to their nests, engage in courtship rituals, and collect the fertilized eggs into their mouths.
after spawning, females leave the male aggregations and live in nursery areas located in shallow water while they incubate the eggs in their mouth and care for the fry  <cit> .
during this period, females become also more aggressive, defending the brood against predators and conspecifics  <cit> .
combining physiological with genomic approaches promises to provide novel insights into how simple social signals in a single sensory channel  are processed in order to generate context-appropriate behavioral responses.
in the present study we therefore first characterize  electrophysiological responses of the olfactory epithelium to different social odors that convey specific information about male social status  and female reproductive state .
we then used a 19 k cichlid microarray platform to analyze the corresponding gene expression profiles in the same individuals in specific brain areas known to be involved in the processing of olfactory information: the olfactory bulb and area dp .
our results show that the olfactory system clearly discriminates stimuli depending on social salience physiologically at the sensory periphery and transcriptionally in central processing centers.
RESULTS
olfactory stimulation
the overall patterns of response to social odors measured with eog recordings  were similar to those previously reported for this species  <cit> .
the mean normalized eog amplitude evoked by subordinate male urine at a dilution of 1: <dig> was significantly smaller  than that elicited by urine samples of dominant males .
furthermore, the mean of normalized responses to water extracts from pre females at a dilution of 1: <dig>  was significantly higher than that from post females .figure  <dig>
olfactory responses of male tilapia to different stimuli.
on the left hand-side, typical electro-olfactograms  recorded in response to different stimuli: in blue – controls for normalization – serine  and blank ; in pink – male urine  – from dominant  and subordinate  males; in light green – extracts of female water  – from pre-ovulatory  and post-ovulatory  females.
on the top-half on right hand side, normalized eog amplitudes  elicited by all stimuli: s ; b ; dom ; sub ; pre ; post ; after 45 min of stimulation .
on the bottom-half, a depiction of the tilapia’s olfactory rosette  and the apparatus for olfactory stimulation and electrophysiological recording of olfactory evoked potentials.
our results show that dom and pre stimuli elicited greater responses than sub or post stimuli, suggesting that males can discriminate social status and reproductive state of social partners based on olfactory cues alone.
the chemical nature of the active odorants which allow for these discriminations is still unknown.
nonetheless, recent work suggests that males can assess a rival’s fighting ability based on the olfactory information present in their urine  <cit> , which might enable them to avoid time consuming and energetically costly escalated fights  <cit>  and thus stabilize social hierarchies  <cit> .
thus, the eog responses measured in the sensory neurons at the olfactory rosette suggest that they are well adapted to discriminate between urinary odorants of different male social status, which might contribute to reduce aggression and escalation of fights in a social context.
moreover, males seem to be able to discriminate between females in different stages of their reproductive cycle, probably due to specific odorants released into the water by pre females, as previously suggested for this species  <cit> .
analysis of gene expression profiles
analysis of ob and dp gene expression revealed hundreds of differently expressed genes after stimulation with any of the four different social stimuli .
considering the initial more than 19 k unique expressed sequence tags  included in the analysis, over 72% hybridized with our samples  in both ob and dp, confirming the usefulness of heterologous hybridization  <cit> .
a bayesian analysis of gene expression levels  <cit>  revealed that  <dig> of the surveyed genes in the ob showed significant differences among the four olfactory stimuli, whereas in dp only  <dig> genes were differentially expressed .
no genes were found to be up- or down-regulated simultaneously in both regions, suggesting that region specific molecular processes are activated by olfactory stimulation and neural transmission.
another interesting observation concerning the number of differently expressed genes in each of these two olfactory processing centers was that at the first relay station, ob, the comparison between male and female cues seems to elicit a considerable surplus of gene regulatory activity, with more than  <dig> genes being differently expressed .
however, at the olfactory pallium  this number decreases substantially and the comparison between pre and post females emerges with almost  <dig> differently expressed genes .table  <dig>
list of all significantly expressed genes and go terms, organized by each one of the four olfactory phenotypes compared for both brain areas tested
ob
dp
olfactory phenotypes compared
differently expressed genes
#features go analysis
sample sizes
differently expressed genes
#features go analysis
sample sizes
table includes differently expressed genes, the number of features annotated considered for the gene ontology analysis and the sample size considered for each phenotype, each comparison and each area sampled.
dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract.
.
unsupervised hierarchical clustering of differentially expressed genes  for all four olfactory stimuli and both brain areas sampled .
on the top right, a sagittal view of a tilapia’s brain cut by two lines  representing the location of the coronal cuts depicted just below illustrating the areas sampled .
bootstrap values are shown on clustergrams.
on the heatmaps, blue represents significantly down-regulated genes, yellow up-regulated genes and black intermediate levels of expression.
confidence values of cluster nodes were calculated using bootstrapping .
olfactory stimuli used in this study: dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract.
brain regions analyzed: olfactory bulb , green box; posterior part of the dorsal telencephalon , purple box.
list of all significantly regulated candidate genes for each one of the four olfactory comparisons made for both brain areas, organized according to presumed functional categories.
on the top, are represented both brain areas sampled , below are presented some significantly expressed target genes according to the chemical categories compared for each region.
green  and red  arrows indicate if genes were over- or under-expressed, respectively.
olfactory stimuli compared: dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract.
a hierarchical cluster of these differently expressed genes in the ob and dp revealed interesting patterns of neuromolecular activity.
in both brain regions, the transcriptional response of males exposed to dom male urine was most similar to that of males exposed to post female water extract, and the transcriptional response to sub male urine was most similar to the response to pre female water extract when we compare all stimuli .
the evidence for olfactory discrimination among stimuli in both brain regions reinforces the idea of a functional organization of the fish olfactory system with parallel pathways flowing from the sensory epithelia via the olfactory bulb into the pallium, conveying specific odor information  <cit> .
furthermore, the different brain regions seem to preferentially process certain stimuli, with sex differences in odors being mainly processed in the ob and subsequent odor differentiation within each sex being processed in area dp.
cummings et al.
<cit>  concluded that these neuromolecular processes drive behavioral responses in the context of female mate choice in swordtails.
unlike olfactory cues in our experiments, female choice in this species activated a suite of genes in response to classes of social stimuli: specific pathways were either up- or down-regulated when females were exposed to males or to other females.
from an ecological point a view, these surprisingly similar transcriptional responses of the ob and dp to sub males and pre females might be explained by the distinctive information conveyed by each behavioral phenotype and by shared valence and salience of their odors.
it is possible that chemical signals emitted by sub males are feminized, which would help to explain why dom males are occasionally observed to direct courtship behavior towards sub males  <cit> .
sub males and pre females shoal together and share the same body coloration.
when courted by dom males sub males exhibit female-like behaviors, which include following the dom male to the spawning pit and getting involved in the full spawning sequence  <cit> .
this behavior allows sub males to remain inside the breeding aggregations where they might attempt sneak fertilizations  <cit> .
despite having mature testes  <cit> , sub males present lower androgen levels  <cit> , lower expression of secondary sex characters  <cit> , and undergo androgen-dependent morphological changes in the urinary bladder and urine storage capacity, reducing its volume to a more female-like size  <cit> , which may also affect the composition of their urine.
the similarity between the gene expression patterns elicited by dom male and post olfactory signals is more difficult to explain.
both social phenotypes are usually very aggressive  <cit> , which might explain some similarities in chemical information.
other possible similarities of the odor bouquet released by these two groups could be related to the significantly reduced food intake these fish experience compared with sub males and pre females, respectively, or the high metabolic rates needed to endure a continuous effort like territorial defense by doms or offspring care by post females  <cit> .
in rodents, for example olfactory sensitivity seems to increase in fasted animals  <cit> , possibly due to a leptin-based modulation of the olfactory mucosa in response to the nutritional status of the animals  <cit> .
this link between food intake and olfaction in rodents could potentially be present in teleosts since it could act as an important eco-ethological adaptation increasing the efficacy of foraging animals when fasted.
the comparison between transcriptional profiles of males stimulated with social olfactory cues with the electrophysiological data gathered from the same males but at the level of the olfactory epithelium also raises some interesting points.
the olfactory epithelium appears to be more sensitive to dom male and pre female stimuli but discrimination between the sexes does not seem to occur at this level of sensory processing .
however, at the level of the ob the gene expression profiles suggest that males have the relevant information available that allows sex discrimination , reinforcing the salience of olfactory cues in social communication in cichlids and teleost fishes in general  <cit> .
since eog recordings and gene expression were collected from the same individuals, we calculated correlation coefficients between the average eog amplitude and the expression levels of the genes that were differentially expressed across the four treatments for the two brain regions studied.
considering a r ≥ consias indicative of an association with a high effect size  <cit> ,  <dig> %  and  <dig> %  of the differentially expressed genes showed high effect size correlations with the eog response in the ob and dp areas, respectively .
the fact that mrna levels of only a minority of genes that were differentially expressed in olfactory processing brain regions correlated with the response of the sensory epithelium might be due to the fact that the eog is an extracellular recording that integrates the overall response of the olfactory mucosa to the stimulus.
therefore, it mainly represents the salience of the stimulus to the fish.
on the other hand, the differentially expressed genes are part of specific signaling pathways that most probably were being activated in response to different dimensions of the stimulus as it was being processed at the different relay stations of the olfactory system.
therefore, the overall lack of association between eog amplitude and gene activity is not surprising.figure  <dig>
hierarchical clustering of significantly different expressed genes  for the comparison of three pairs of olfactory cues in both brain areas sampled .
green box: olfactory bulb  expression; purple box: posterior part of the dorsal telencephalon .
left panels: comparison of female  and male  cues independent of status or condition; middle panels, comparison of pre-  and post-  ovulatory female cues; right panels: comparison of dominant and subordinate male cues.
the heatmaps  show estimated gene expression levels.
confidence values of cluster nodes were calculated using bootstrapping.
go analysis
our gene ontology analysis allowed us to categorize differentially expressed genes in relation to the molecular functions, biological processes, and cellular components they are associated with .
although the results of go analyses can be difficult to interpret, they provide a framework for developing novel hypotheses that could potentially inform new approaches to the molecular underpinnings of socially regulated brain function  <cit> .
we therefore asked whether any go categories are under- or over-represented in one stimulus condition compared to the others.
in all comparisons analyzed , go terms could be applied to more than 55% of the regulated array features.
interestingly, the functional categories expressing enriched pathways with extreme over- and under-representation are also more numerous for the distinction between males and females in the ob, and rather scarce for the same comparison at the dp level.
in the latter, the number of enriched go terms is smaller and more evenly distributed among the remaining comparisons .
this suggests that already at the ob level, the first relay station in the olfactory circuit, information on the sex of a nearby conspecific might be filtered out, which in a social interaction would be reinforced by visual cues ascertaining this information and triggering the appropriate behavioral response.figure  <dig>
gene ontology  analysis summary for each one of the four olfactory comparisons made for both brain areas.
under-represented categories are shown in shades of blue and over-represented ones in shades of red .
the different go vocabularies are shown separately: cellular component, biological process and molecular function; along with the p-values  and go names and numbers .
dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract.
candidate genes
besides activating specific molecular pathways, a number of candidate genes are also significantly regulated in one of the two brain areas sampled from dominant males stimulated with different social odors in this experiment.
somatotropin, a member of the growth hormone  family, is significantly up-regulated in the ob  in response to either the odor of a dom male or the odor of females .
on the other hand, in dp somatostatin, a known gh production inhibitor, is down-regulated after stimulation with dom scent.
regulation of these members of the gh signaling are usually related to differential growth, a characteristically plastic trait in cichlids in response to changes in the social environment  <cit> .
somatostatin is known to play an important role in the complex interplay between social behavior and somatic growth in cichlid fishes  <cit> , likely regulating the allocation of energetic resources between reproduction and growth  <cit> .
somatostatin down-regulation only in response to the presence of an odor cue of a potentially threatening high-ranked male along with the up-regulation of somatotropin, suggests the preparation for the physical strain involved in an approaching agonistic interaction.
other candidate genes were also up-regulated in stimulated dominant males, such as: brain aromatase in the ob and gonadotropin-releasing hormone  and pro-opiomelanocortin alpha  <dig>  in dp.
it has been previously showed in a. burtoni that pomc α <dig> is more highly expressed in the brain of dom males compared with subs  <cit> , and α-melanocyte stimulating hormone  has been shown to stimulate dom traits such as aggression and coloration  <cit> .
interestingly, pomc α <dig> exhibits a complex expression pattern through brain and peripheral tissues of in a. burtoni  <cit> , which makes this gene a prime candidate for future studies.
the up-regulation of gnrh <dig> after an olfactory stimulation with sub male odor reinforces the idea of a putative feminization of their urine discussed above, since gnrh integrates the animal’s internal physiological state with incoming external cues to regulate reproduction in males.
in cichlid fish, reproductive status influences the regulation of this neuropeptide and seasonal fluctuations of gnrh receptor levels in the brain can modulate olfactory processing, regulating the animal’s plasticity in olfactory responsiveness  <cit> .
importantly, gnrh receptors are expressed in both ob and dp of the nile tilapia, oreochromis niloticus  <cit> .
interestingly, gnrh up-regulation in an extra-hypothalamic area, like dp, can also be found in rats, where gnrh mrna is present both in the olfactory piriform cortex  and in the olfactory bulb  <cit> .
egr- <dig> and cytochrome c oxidase  were both down-regulated in ob of males stimulated with male social odor when compared with female social odor.
both genes are known markers of neural activity  <cit>  and the regulation of egr- <dig> appears to have a pivotal role in recruiting specific neural pathways required for long-term memory processes  <cit> .
egr-1-deficient mice seem to be unable to form long-term memories in behavioral tasks, such as olfactory discrimination, while their short-term memory and early-ltp remain intact  <cit> .
in zebrafish, egr- <dig> activity seems to be involved in imprinting processes in early life stages and later in kin recognition, especially in the ob, since rather low basal expression levels are found in the dp  <cit> .
in summary, egr- <dig> down-regulation in the ob of dom males in response to olfactory cues of male conspecifics, suggests a possible role of olfactory modulation on memory consolidation of social odors, but on the other hand, little is known about cox modulation with olfactory social stimuli.
in another cichlid species, astatotilapia burtoni, when males were presented with visual and olfactory signals, each sensory modality was sufficient to elicit an androgen response in an intruder challenge paradigm, but chemical stimulation alone did not induce the immediate-early gene c-fos, another marker of neuronal activity  <cit> , in the brain  <cit> .
CONCLUSIONS
in the present study we have used a transcriptome-scale analysis of the molecular systems regulated by social olfactory experience in order to investigate the proximate mechanisms underlying olfactory stimulation.
we found that dom males stimulated with different socially salient chemical cues exhibited some degree of discrimination between stimuli in the olfactory epithelium.
also, different salient olfactory stimuli resulted in considerable variation in ob and dp gene expression profiles of dom males, suggesting that the olfactory system can discriminate social status and reproductive condition, as well as, its sex based solely on its chemical signature.
our findings also underscore the extensive transcriptional plasticity in response to the social environment and reinforce the importance of uncovering the molecular and cellular factors and constraints governing olfactory function and the neurogenomic consequences of experiencing different social olfactory cues.
these different neurogenomic states likely modulate and optimize behavior according to social context  <cit> .
future comparative studies focusing on the neuroplasticity underlying the diverse behavioral adaptations found in cichlids will help us understand the processes by which this teleost family has diversified so rapidly.

 group living animals have to adjust the expression of social behavior to the nuances of daily social life and to transitions between life-history stages, and their ability to do so impacts on their darwinian fitness. this socially driven behavioral plasticity induces changes in brain neurogenomic states that underlie different behavioral repertoires. thus, reprogramming the transcriptome in response to the social environment allows an animal to switch between adaptive behavioral states. gene expression profiling enables the study of this dynamic relationship between genotype and behavior and to unveil the genetic networks behind complex behaviors. in addition, the development of whole-genome sequencing, microarrays and other genomic resources for non-traditional model organisms, but with complex social repertoires, has provided relevant insights on how complex genotypes are translated to produce meaningful behaviors. in recent years, numerous studies have described the influence of social environment and of social interactions on transcriptional and neural activity. for example, caste differentiation in the honey bee, a key feature in eusocial insects, is influenced not only by heritable traits but also by variations in the regulation of molecular pathways linked with several life-history traits, such as nutrition, metabolism, and reproduction. the activity of aggression-related genes in this species also seems to be under both inherited and environmental influences, varying with age, exposure to alarm-cues and depending on colony environment. the study of gene expression signatures of life history transitions has also been a focus in teleost fishes. for example, life history traits of salmonids have also been addressed in a number of studies showing variation in brain expression profiles related with alternative reproductive and migratory tactics and their interaction with the rearing environment. all the results on the impact of the social environment on the transcriptome highlight new possibilities concerning how social stimuli, as well as more complex interactions between conspecifics, can influence and shape gene translation into producing appropriate behavioral responses, according to external and internal cues and also to the animals’ past experience. most of the studies discussed above characterize fixed and irreversible behavioral phenotypes, which correspond to switches between “static” neurogenomic states. but the interaction between the genome and the environment is also expected to be present in shorter time frames and to be reversible in order to accommodate labile and transient changes in behavioral states in order for flexible adaptive behavior to evolve. behaviorally, a single interaction may have consequences for the performance of the individuals and the outcome of future interactions, but its impact on the neurogenomic state of the individuals has been scarcely characterized. animals integrate sensory information with internal physiology into context-appropriate behavior that ultimately promotes fitness. yet how the brain integrates different sensory modalities in these social contexts remains unclear. for example, presented males of the model cichlid astatotilapia burtoni with sensory information in three social contexts: intruder challenge, reproductive opportunity and a socially neutral situation. the authors found that, compared to the neutral context, a visual stimulus was necessary and sufficient for an aggressive response, whereas chemical and visual stimuli presented alone were sufficient for an androgen response. interestingly, the immediate-early gene c-fos, a neural activity marker, was induced in response to a visual challenge stimulus specifically in dopaminergic neurons of area vc, a putative striatal homologue, whereas presentation of a chemical stimulus alone did not induce c-fos expression in the intruder challenge context. clearly, these results suggest that socially salient sensory cues are processed in a modality-dependent manner in the brain. however, this study did not examine neuromolecular responses of forebrain regions associated with olfactory processing, such as the olfactory bulb or area dp. the mozambique tilapia, oreochromis mossambicus, is an african cichlid fish that has become a model system in the study of neuroendocrine mechanisms underlying socially mediated behavioral changes. the importance of chemical signaling of male social status has been described in this species, and the olfactory system, from sensory epithelium to bulbar and extrabulbar projections, has been well characterized. in nature, o. mossambicus males establish contiguous display territories, which females visit in order to obtain matings. the repertoire of social behavior is highly complex and multimodal, including visual. importantly, male tilapia store urine in their bladders which they use to signal social rank during agonistic interactions with other males or in the presence of pre-ovulatory females. furthermore, males are able to modulate their rate of urination depending on the social environment. an increase of males’ urination rate is observed during agonistic encounters or in the presence of pre-ovulatory females. furthermore, both the volume of stored urine and its olfactory potency, as measured by electro-olfactogram recordings, is higher in dom than in sub males. females do not store urine and have a higher frequency of urination. additionally, females have smaller kidneys, smaller urinary bladders and the urothelial thickness of the inner surface of the bladder is also smaller than in males. finally, the odor of pre-ovulatory females elicits higher amplitude eog responses in males than that of post-ovulatory females. depending on the social environment, tilapia males can exhibit two distinct behavioral phenotypes: dominants and subordinate. dom individuals adopt a typical velvet black coloration and establish breeding territories on the bottom, where they dig nests to which they attract females using courtship displays. sub males present a pale silver coloration and either move around among the breeding territories of dom males or shoal together with females, while they wait for their opportunity for social ascension. sneaking fertilization attempts by sub males have also been reported. changes between these social phenotypes have been shown to activate a cascade of molecular processes and a variety of neuroendocrine pathways which include neuropeptides and steroid hormones. ovulated females visit male breeding arenas when ready to spawn and follow courting males to their nests, engage in courtship rituals, and collect the fertilized eggs into their mouths. after spawning, females leave the male aggregations and live in nursery areas located in shallow water while they incubate the eggs in their mouth and care for the fry. during this period, females become also more aggressive, defending the brood against predators and conspecifics. combining physiological with genomic approaches promises to provide novel insights into how simple social signals in a single sensory channel are processed in order to generate context-appropriate behavioral responses. in the present study we therefore first characterize electrophysiological responses of the olfactory epithelium to different social odors that convey specific information about male social status and female reproductive state. we then used a 19 k cichlid microarray platform to analyze the corresponding gene expression profiles in the same individuals in specific brain areas known to be involved in the processing of olfactory information: the olfactory bulb and area dp. our results show that the olfactory system clearly discriminates stimuli depending on social salience physiologically at the sensory periphery and transcriptionally in central processing centers.  olfactory stimulation the overall patterns of response to social odors measured with eog recordings were similar to those previously reported for this species. the mean normalized eog amplitude evoked by subordinate male urine at a dilution of 1: was significantly smaller than that elicited by urine samples of dominant males. furthermore, the mean of normalized responses to water extracts from pre females at a dilution of 1: was significantly higher than that from post females.figure olfactory responses of male tilapia to different stimuli. on the left hand-side, typical electro-olfactograms recorded in response to different stimuli: in blue – controls for normalization – serine and blank; in pink – male urine – from dominant and subordinate males; in light green – extracts of female water – from pre-ovulatory and post-ovulatory females. on the top-half on right hand side, normalized eog amplitudes elicited by all stimuli: s; b; dom; sub; pre; post; after 45 min of stimulation. on the bottom-half, a depiction of the tilapia’s olfactory rosette and the apparatus for olfactory stimulation and electrophysiological recording of olfactory evoked potentials. our results show that dom and pre stimuli elicited greater responses than sub or post stimuli, suggesting that males can discriminate social status and reproductive state of social partners based on olfactory cues alone. the chemical nature of the active odorants which allow for these discriminations is still unknown. nonetheless, recent work suggests that males can assess a rival’s fighting ability based on the olfactory information present in their urine, which might enable them to avoid time consuming and energetically costly escalated fights and thus stabilize social hierarchies. thus, the eog responses measured in the sensory neurons at the olfactory rosette suggest that they are well adapted to discriminate between urinary odorants of different male social status, which might contribute to reduce aggression and escalation of fights in a social context. moreover, males seem to be able to discriminate between females in different stages of their reproductive cycle, probably due to specific odorants released into the water by pre females, as previously suggested for this species. analysis of gene expression profiles analysis of ob and dp gene expression revealed hundreds of differently expressed genes after stimulation with any of the four different social stimuli. considering the initial more than 19 k unique expressed sequence tags included in the analysis, over 72% hybridized with our samples in both ob and dp, confirming the usefulness of heterologous hybridization. a bayesian analysis of gene expression levels revealed that of the surveyed genes in the ob showed significant differences among the four olfactory stimuli, whereas in dp only genes were differentially expressed. no genes were found to be up- or down-regulated simultaneously in both regions, suggesting that region specific molecular processes are activated by olfactory stimulation and neural transmission. another interesting observation concerning the number of differently expressed genes in each of these two olfactory processing centers was that at the first relay station, ob, the comparison between male and female cues seems to elicit a considerable surplus of gene regulatory activity, with more than genes being differently expressed. however, at the olfactory pallium this number decreases substantially and the comparison between pre and post females emerges with almost differently expressed genes.table list of all significantly expressed genes and go terms, organized by each one of the four olfactory phenotypes compared for both brain areas tested ob dp olfactory phenotypes compared differently expressed genes #features go analysis sample sizes differently expressed genes #features go analysis sample sizes table includes differently expressed genes, the number of features annotated considered for the gene ontology analysis and the sample size considered for each phenotype, each comparison and each area sampled. dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract.. unsupervised hierarchical clustering of differentially expressed genes for all four olfactory stimuli and both brain areas sampled. on the top right, a sagittal view of a tilapia’s brain cut by two lines representing the location of the coronal cuts depicted just below illustrating the areas sampled. bootstrap values are shown on clustergrams. on the heatmaps, blue represents significantly down-regulated genes, yellow up-regulated genes and black intermediate levels of expression. confidence values of cluster nodes were calculated using bootstrapping. olfactory stimuli used in this study: dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract. brain regions analyzed: olfactory bulb, green box; posterior part of the dorsal telencephalon, purple box. list of all significantly regulated candidate genes for each one of the four olfactory comparisons made for both brain areas, organized according to presumed functional categories. on the top, are represented both brain areas sampled, below are presented some significantly expressed target genes according to the chemical categories compared for each region. green and red arrows indicate if genes were over- or under-expressed, respectively. olfactory stimuli compared: dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract. a hierarchical cluster of these differently expressed genes in the ob and dp revealed interesting patterns of neuromolecular activity. in both brain regions, the transcriptional response of males exposed to dom male urine was most similar to that of males exposed to post female water extract, and the transcriptional response to sub male urine was most similar to the response to pre female water extract when we compare all stimuli. the evidence for olfactory discrimination among stimuli in both brain regions reinforces the idea of a functional organization of the fish olfactory system with parallel pathways flowing from the sensory epithelia via the olfactory bulb into the pallium, conveying specific odor information. furthermore, the different brain regions seem to preferentially process certain stimuli, with sex differences in odors being mainly processed in the ob and subsequent odor differentiation within each sex being processed in area dp. cummings et al. concluded that these neuromolecular processes drive behavioral responses in the context of female mate choice in swordtails. unlike olfactory cues in our experiments, female choice in this species activated a suite of genes in response to classes of social stimuli: specific pathways were either up- or down-regulated when females were exposed to males or to other females. from an ecological point a view, these surprisingly similar transcriptional responses of the ob and dp to sub males and pre females might be explained by the distinctive information conveyed by each behavioral phenotype and by shared valence and salience of their odors. it is possible that chemical signals emitted by sub males are feminized, which would help to explain why dom males are occasionally observed to direct courtship behavior towards sub males. sub males and pre females shoal together and share the same body coloration. when courted by dom males sub males exhibit female-like behaviors, which include following the dom male to the spawning pit and getting involved in the full spawning sequence. this behavior allows sub males to remain inside the breeding aggregations where they might attempt sneak fertilizations. despite having mature testes, sub males present lower androgen levels, lower expression of secondary sex characters, and undergo androgen-dependent morphological changes in the urinary bladder and urine storage capacity, reducing its volume to a more female-like size, which may also affect the composition of their urine. the similarity between the gene expression patterns elicited by dom male and post olfactory signals is more difficult to explain. both social phenotypes are usually very aggressive, which might explain some similarities in chemical information. other possible similarities of the odor bouquet released by these two groups could be related to the significantly reduced food intake these fish experience compared with sub males and pre females, respectively, or the high metabolic rates needed to endure a continuous effort like territorial defense by doms or offspring care by post females. in rodents, for example olfactory sensitivity seems to increase in fasted animals, possibly due to a leptin-based modulation of the olfactory mucosa in response to the nutritional status of the animals. this link between food intake and olfaction in rodents could potentially be present in teleosts since it could act as an important eco-ethological adaptation increasing the efficacy of foraging animals when fasted. the comparison between transcriptional profiles of males stimulated with social olfactory cues with the electrophysiological data gathered from the same males but at the level of the olfactory epithelium also raises some interesting points. the olfactory epithelium appears to be more sensitive to dom male and pre female stimuli but discrimination between the sexes does not seem to occur at this level of sensory processing. however, at the level of the ob the gene expression profiles suggest that males have the relevant information available that allows sex discrimination, reinforcing the salience of olfactory cues in social communication in cichlids and teleost fishes in general. since eog recordings and gene expression were collected from the same individuals, we calculated correlation coefficients between the average eog amplitude and the expression levels of the genes that were differentially expressed across the four treatments for the two brain regions studied. considering a r ≥ consias indicative of an association with a high effect size, % and % of the differentially expressed genes showed high effect size correlations with the eog response in the ob and dp areas, respectively. the fact that mrna levels of only a minority of genes that were differentially expressed in olfactory processing brain regions correlated with the response of the sensory epithelium might be due to the fact that the eog is an extracellular recording that integrates the overall response of the olfactory mucosa to the stimulus. therefore, it mainly represents the salience of the stimulus to the fish. on the other hand, the differentially expressed genes are part of specific signaling pathways that most probably were being activated in response to different dimensions of the stimulus as it was being processed at the different relay stations of the olfactory system. therefore, the overall lack of association between eog amplitude and gene activity is not surprising.figure hierarchical clustering of significantly different expressed genes for the comparison of three pairs of olfactory cues in both brain areas sampled. green box: olfactory bulb expression; purple box: posterior part of the dorsal telencephalon. left panels: comparison of female and male cues independent of status or condition; middle panels, comparison of pre- and post- ovulatory female cues; right panels: comparison of dominant and subordinate male cues. the heatmaps show estimated gene expression levels. confidence values of cluster nodes were calculated using bootstrapping. go analysis our gene ontology analysis allowed us to categorize differentially expressed genes in relation to the molecular functions, biological processes, and cellular components they are associated with. although the results of go analyses can be difficult to interpret, they provide a framework for developing novel hypotheses that could potentially inform new approaches to the molecular underpinnings of socially regulated brain function. we therefore asked whether any go categories are under- or over-represented in one stimulus condition compared to the others. in all comparisons analyzed, go terms could be applied to more than 55% of the regulated array features. interestingly, the functional categories expressing enriched pathways with extreme over- and under-representation are also more numerous for the distinction between males and females in the ob, and rather scarce for the same comparison at the dp level. in the latter, the number of enriched go terms is smaller and more evenly distributed among the remaining comparisons. this suggests that already at the ob level, the first relay station in the olfactory circuit, information on the sex of a nearby conspecific might be filtered out, which in a social interaction would be reinforced by visual cues ascertaining this information and triggering the appropriate behavioral response.figure gene ontology analysis summary for each one of the four olfactory comparisons made for both brain areas. under-represented categories are shown in shades of blue and over-represented ones in shades of red. the different go vocabularies are shown separately: cellular component, biological process and molecular function; along with the p-values and go names and numbers. dom- dominant male urine; sub- subordinate male urine; pre- pre-ovulatory female water extract; post- post-ovulatory female water extract. candidate genes besides activating specific molecular pathways, a number of candidate genes are also significantly regulated in one of the two brain areas sampled from dominant males stimulated with different social odors in this experiment. somatotropin, a member of the growth hormone family, is significantly up-regulated in the ob in response to either the odor of a dom male or the odor of females. on the other hand, in dp somatostatin, a known gh production inhibitor, is down-regulated after stimulation with dom scent. regulation of these members of the gh signaling are usually related to differential growth, a characteristically plastic trait in cichlids in response to changes in the social environment. somatostatin is known to play an important role in the complex interplay between social behavior and somatic growth in cichlid fishes, likely regulating the allocation of energetic resources between reproduction and growth. somatostatin down-regulation only in response to the presence of an odor cue of a potentially threatening high-ranked male along with the up-regulation of somatotropin, suggests the preparation for the physical strain involved in an approaching agonistic interaction. other candidate genes were also up-regulated in stimulated dominant males, such as: brain aromatase in the ob and gonadotropin-releasing hormone and pro-opiomelanocortin alpha in dp. it has been previously showed in a. burtoni that pomc α is more highly expressed in the brain of dom males compared with subs, and α-melanocyte stimulating hormone has been shown to stimulate dom traits such as aggression and coloration. interestingly, pomc α exhibits a complex expression pattern through brain and peripheral tissues of in a. burtoni, which makes this gene a prime candidate for future studies. the up-regulation of gnrh after an olfactory stimulation with sub male odor reinforces the idea of a putative feminization of their urine discussed above, since gnrh integrates the animal’s internal physiological state with incoming external cues to regulate reproduction in males. in cichlid fish, reproductive status influences the regulation of this neuropeptide and seasonal fluctuations of gnrh receptor levels in the brain can modulate olfactory processing, regulating the animal’s plasticity in olfactory responsiveness. importantly, gnrh receptors are expressed in both ob and dp of the nile tilapia, oreochromis niloticus. interestingly, gnrh up-regulation in an extra-hypothalamic area, like dp, can also be found in rats, where gnrh mrna is present both in the olfactory piriform cortex and in the olfactory bulb. egr- and cytochrome c oxidase were both down-regulated in ob of males stimulated with male social odor when compared with female social odor. both genes are known markers of neural activity and the regulation of egr- appears to have a pivotal role in recruiting specific neural pathways required for long-term memory processes. egr-1-deficient mice seem to be unable to form long-term memories in behavioral tasks, such as olfactory discrimination, while their short-term memory and early-ltp remain intact. in zebrafish, egr- activity seems to be involved in imprinting processes in early life stages and later in kin recognition, especially in the ob, since rather low basal expression levels are found in the dp. in summary, egr- down-regulation in the ob of dom males in response to olfactory cues of male conspecifics, suggests a possible role of olfactory modulation on memory consolidation of social odors, but on the other hand, little is known about cox modulation with olfactory social stimuli. in another cichlid species, astatotilapia burtoni, when males were presented with visual and olfactory signals, each sensory modality was sufficient to elicit an androgen response in an intruder challenge paradigm, but chemical stimulation alone did not induce the immediate-early gene c-fos, another marker of neuronal activity, in the brain.  in the present study we have used a transcriptome-scale analysis of the molecular systems regulated by social olfactory experience in order to investigate the proximate mechanisms underlying olfactory stimulation. we found that dom males stimulated with different socially salient chemical cues exhibited some degree of discrimination between stimuli in the olfactory epithelium. also, different salient olfactory stimuli resulted in considerable variation in ob and dp gene expression profiles of dom males, suggesting that the olfactory system can discriminate social status and reproductive condition, as well as, its sex based solely on its chemical signature. our findings also underscore the extensive transcriptional plasticity in response to the social environment and reinforce the importance of uncovering the molecular and cellular factors and constraints governing olfactory function and the neurogenomic consequences of experiencing different social olfactory cues. these different neurogenomic states likely modulate and optimize behavior according to social context. future comparative studies focusing on the neuroplasticity underlying the diverse behavioral adaptations found in cichlids will help us understand the processes by which this teleost family has diversified so rapidly. 
we then performed a genome-scale gene expression analysis of the olfactory bulb and the olfactory cortex homolog in order to identify the neuromolecular systems involved in processing these social stimuli. this species has a rich repertoire of social behaviors during which both visual and chemical information are conveyed to conspecifics. specifically, dominant males increase their urination frequency during agonist encounters and during courtship to convey chemical information reflecting their dominance status. independent of the proximate mechanisms, at the neuromolecular level social plasticity relies on the regulation of gene expression, such that different neurogenomic states emerge in response to different social stimuli and the switches between states are orchestrated by signaling pathways that interface the social environment and the genotype. we recorded electro-olfactograms to test the extent to which the olfactory epithelium can discriminate between olfactory information from dominant and subordinate males as well as from pre- and post-spawning females. here, we test this hypothesis by characterizing the changes in the brain profile of gene expression in response to social odors in the mozambique tilapia, oreochromis mossambicus. our results show that different olfactory stimuli from conspecifics’ have a major impact in the brain transcriptome, with different chemical social cues eliciting specific patterns of gene expression in the brain. these results confirm the role of rapid changes in gene expression in the brain as a genomic mechanism underlying behavioral plasticity and reinforce the idea of an extensive transcriptional plasticity of cichlid genomes, especially in response to rapid changes in their social environment. animals adjust the expression of their social behavior to the daily changes in social life and to transitions between life-history stages, and this ability has an impact in their darwinian fitness. this behavioral plasticity may be achieved either by rewiring or by biochemically switching nodes of the neural network underlying social behavior in response to perceived social information.
 social plasticity is a pervasive feature of animal behavior. animals adjust the expression of their social behavior to the daily changes in social life and to transitions between life-history stages, and this ability has an impact in their darwinian fitness. this behavioral plasticity may be achieved either by rewiring or by biochemically switching nodes of the neural network underlying social behavior in response to perceived social information. independent of the proximate mechanisms, at the neuromolecular level social plasticity relies on the regulation of gene expression, such that different neurogenomic states emerge in response to different social stimuli and the switches between states are orchestrated by signaling pathways that interface the social environment and the genotype. here, we test this hypothesis by characterizing the changes in the brain profile of gene expression in response to social odors in the mozambique tilapia, oreochromis mossambicus. this species has a rich repertoire of social behaviors during which both visual and chemical information are conveyed to conspecifics. specifically, dominant males increase their urination frequency during agonist encounters and during courtship to convey chemical information reflecting their dominance status.  we recorded electro-olfactograms to test the extent to which the olfactory epithelium can discriminate between olfactory information from dominant and subordinate males as well as from pre- and post-spawning females. we then performed a genome-scale gene expression analysis of the olfactory bulb and the olfactory cortex homolog in order to identify the neuromolecular systems involved in processing these social stimuli.  our results show that different olfactory stimuli from conspecifics’ have a major impact in the brain transcriptome, with different chemical social cues eliciting specific patterns of gene expression in the brain. these results confirm the role of rapid changes in gene expression in the brain as a genomic mechanism underlying behavioral plasticity and reinforce the idea of an extensive transcriptional plasticity of cichlid genomes, especially in response to rapid changes in their social environment. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cichlidolfactionolfactory bulbtelencephalonmicroarraytranscriptomicsissue-copyright-statement© the author 2015 