Project description:Purpose: The goal of this study is to identify genes that showed a pattern of differential expression between species within activated neurons during mirror-elicited aggression between two species of Lake Malawi cichlid fish which differ in aggressive behaviors, Mchenga conophoros (MC, sand-dwelling) and Petrotilapia chitimba (PC, rock-dwelling). To do this, we use antibodies to phosphorylated ribosomal protein S6 (pS6) to enrich for transcripts bound to phosphorylated ribosomes followed by RNA-seq (PhosphoTRAP). In neurons, this phosphorylation occurs downstream of the binding of neurotransmitters and, thus, pS6 antibodies are increasingly being used as markers of neural activity, similar to immediate early genes (IEGs) like c-fos or egr-1. Methods: Whole brain mRNA samples were collected from 8 adult male cichlids (4 MC, 4 PC) 90min following either 15min exposure to mirror in home tank (Mirror Test) or a Control condition. RNA-immunoprecipitation using antibodies to pS6 were performed and RNA was extracted from immunoprecipitated (IP) RNA samples. We also collected total RNA (input), prior to immunoprecipitation. For details see extraction protocol below. RNA from both input and IP samples was reverse transcribed using the SMART-Seq v4 Ultra Low Input RNA kit. Libraries were prepped using the NEBNext Ultra II FS DNA library Prep Kit for Illumina and sequenced. Sequence reads that passed quality filters were aligned to the Metriaclima zebra (Malawi cichlid) reference genome UMD2A using HiSat2. Transcripts were quantified using StringTie and differential expressional analyses were performed using DESeq2. Results: We identified a large number of genes showing differential expression in mirror-elicited aggression, as well as many genes that differ between ecotypes. These genes, which may underly species differences in behavior, include several neuropeptides, genes involved in the synthesis of steroid hormones, and neurotransmitter activity. Conclusions: This work lays the foundation for future experiments using this emerging genetic model system to investigate the genomic basis of evolved species differences in both brain and behavior.

Project description:Neural crest (NC) is a vertebrate-specific embryonic progenitor cell population at the basis of important vertebrate features such as the craniofacial skeleton and pigmentation patterns. Despite the wide-ranging variation of NC-derived traits across vertebrates, the contribution of NC to species diversification remains underexplored. Here, leveraging the adaptive diversity of African Great Lakes' cichlid species, we combined comparative transcriptomics and population genomics to investigate the evolution of the NC genetic program in the context of their morphological divergence. Our analysis revealed substantial differences in transcriptional landscapes across somitogenesis, an embryonic period coinciding with NC development and migration. This included dozens of genes with described functions in the vertebrate NC gene regulatory network, several of which showed signatures of positive selection. Among candidates showing between-species expression divergence, we focused on teleost-specific paralogs of the NC-specifier sox10 (sox10a and sox10b) as prime candidates to influence NC development. These genes, expressed in NC cells, displayed remarkable spatio-temporal variation in cichlids, suggesting their contribution to interspecific morphological differences, such as craniofacial structures and pigmentation. Finally, through CRISPR/Cas9 mutagenesis, we demonstrated the functional divergence between cichlid sox10 paralogs, with the acquisition of a novel skeletogenic function by sox10a. When compared with teleost models zebrafish and medaka, our findings reveal that sox10 duplication, although retained in most teleost lineages, had variable functional fates across their phylogeny. Altogether, our study suggests that NC-related processes—particularly those controlled by sox10s—are involved in generating morphological diversification between species and lays the groundwork for further investigations into the mechanisms underpinning vertebrate NC diversification.

Project description:Aggression is ubiquitous among social species and functions to maintains social dominance hierarchies. The African cichlid fish Astatotilapia burtoni is an ideal study species for studying aggression due to their unique and flexible dominance hierarchy. However, female aggression in this species and the neural mechanisms of aggression in both sexes is not well understood. To further understand the potential sex differences in aggression in this species, we characterized aggression in male and female A. burtoni in a mirror assay. We then quantified neural activation patterns in brain regions of the social behavior network (SBN) to investigate if differences in behavior are reflected in the brain with immunohistochemistry by detecting the phosphorylated ribosome marker phospho-S6 ribosomal protein (pS6), a marker for neural activation. We found that A. burtoni perform both identical and sex-specific aggressive behaviors in response to a mirror assay. We observed sex differences in pS6 immunoreactivity in the Vv, a homolog of the lateral septum in mammals. Males but not females had higher ps6 immunoreactivity in the ATn after the aggression assay. The ATn is a homolog of the ventromedial hypothalamus in mammals, which is strongly implicated in the regulation of aggression in males. Several regions also have higher pS6 immunoreactivity in negative controls than fish exposed to a mirror, implicating a role for inhibitory neurons in suppressing aggression until a relevant stimulus is present. Male and female A. burtoni display both similar and sexually dimorphic behavioral patterns in aggression in response to a mirror assay. There are also sex differences in the corresponding neural activation patterns in the SBN. In mirror males but not females, the ATn clusters with the POA, revealing a functional connectivity of these regions that is triggered in an aggressive context in males. These findings suggest that distinct neural circuitry underlie aggressive behavior in male and female A. burtoni, serving as a foundation for future work investigating the molecular and neural underpinnings of sexually dimorphic behaviors in this species to reveal fundamental insights into understanding aggression.

Project description:Aggression is ubiquitous among social species and can function to maintain social dominance hierarchies. The African cichlid fish Astatotilapia burtoni is an ideal study species for studying aggression due to their dominance hierarchy and robust behavioral repertoire. To further understand the potential sex differences in aggression in this species, we characterized aggression in male and female A. burtoni in a mirror assay. We then quantified neural activation patterns in brain regions of the social behavior network (SBN) to investigate if differences in behavior are reflected in the brain with immunohistochemistry by detecting the phosphorylated ribosome marker phospho-S6 ribosomal protein (pS6), a marker for neural activation. We found that A. burtoni perform both identical and sex-specific aggressive behaviors in response to a mirror assay. Females had greater pS6 immunoreactivity than males in the Vv (ventral part of the ventral telencephalon), a homolog of the lateral septum in mammals. Males but not females had higher pS6 immunoreactivity in the ATn after the aggression assay. The ATn (anterior tuberal nucleus) is a homolog of the ventromedial hypothalamus in mammals, which is strongly implicated in the regulation of aggression in males. Several regions also have higher pS6 immunoreactivity in negative controls than fish exposed to a mirror, implicating a role for inhibitory neural processes in suppressing aggression until a relevant stimulus is present. Male and female A. burtoni display both similar and different behavioral patterns in aggression in response to a mirror assay. There are also sex differences in the corresponding neural activation patterns in the SBN. In mirror males but not females, the ATn clusters with the POA, revealing a functional connectivity of these regions that is triggered in an aggressive context in males. These findings suggest that distinct neural circuitry underlie aggressive behavior in male and female A. burtoni, serving as a foundation for future work investigating the molecular and neural underpinnings of sex differences in behavior in this species to reveal fundamental insights into understanding aggression.

Project description: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 the ability to change in these ways impacts their Darwinian fitness. This behavioral plasticity may be achieved either by rewiring or by biochemically switching nodes of the neural network underlying the 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 conspecificsM-bM-^@M-^Y 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 behavioural plasticity and reinforce the idea of an extensive transcriptional plasticity of cichlid genomes, especially in response to rapid changes in their social environment. Brain samples from 40 African cichlid males, Oreochromis mossambicus were collected after stimulation with different social olfactory stimuli. Samples were collected from 2 brain areas: BO and Dp after males were exposed to dominant (DOM) and subordinate (SUB) male urine and pre- (PRE) and post-ovulatory (POST) female scent. In OB 5 replicates were collected from males exposed to DOM and 6 to the other stimuli. For Dp 5 replicates were collected from males exposed to DOM and POST, 4 to SUB and 6 to PRE.

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Ecotype differences in aggression, neural activity and behaviorally relevant gene expression in cichlid fish

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