Neural activation in the olfactory epithelium of East African cichlid in response to odorant exposure

Fish use olfaction to gain a variety of information. To know what they receive and how they receive is important for understanding the species. However, studies on fish olfactory or pheromone receptors are still few and most of them are based on the neural response from a cultured cell. Here, we established a method to detect a biological-derived neural response from the olfactory epithelium of East African cichlid, the most diversified fish lineage, by c-fos in situ hybridization. We tested the response of microvillous neurons, which are expected to be dominated by V2R-expressing neurons, against several odorants. We showed microvillous neurons respond to amino acids the most whereas they do not respond to conjugated steroids. We next tested the response of V2R receptors. Although some subfamilies of V2R responded to amino acids, other did not respond which contrasts with the traditional hypothesis. Especially, one V2R subfamily responded to arginine. Not all the copies in this subfamily responded to arginine, which indicates the ligand differentiation in the cichlid-specifically expanded subfamily. Finally, we tested the response of putative pheromone receptor V1R to male urine. For this experiment, we established a new method to collect urine from cichlid. We showed two V1R receptors responded to male urine. Moreover, we showed that V1R2 receptor responded to 4-hydroxyphenyl acetate and lithocholic acid. This study is expected to provide a basis for the study on the olfaction of East African cichlids.

Evolution of regulatory networks associated with traits under selection in cichlids

Background Seminal studies of vertebrate protein evolution speculated that gene regulatory changes can drive anatomical innovations. However, very little is known about gene regulatory network (GRN) evolution associated with phenotypic effect across ecologically diverse species. Here we use a novel approach for comparative GRN analysis in vertebrate species to study GRN evolution in representative species of the most striking examples of adaptive radiations, the East African cichlids. We previously demonstrated how the explosive phenotypic diversification of East African cichlids can be attributed to diverse molecular mechanisms, including accelerated regulatory sequence evolution and gene expression divergence. Results To investigate these mechanisms across species at a genome-wide scale, we develop a novel computational pipeline that predicts regulators for co-extant and ancestral co-expression modules along a phylogeny, and candidate regulatory regions associated with traits under selection in cichlids. As a case study, we apply our approach to a well-studied adaptive trait—the visual system—for which we report striking cases of network rewiring for visual opsin genes, identify discrete regulatory variants, and investigate their association with cichlid visual system evolution. In regulatory regions of visual opsin genes, in vitro assays confirm that transcription factor binding site mutations disrupt regulatory edges across species and segregate according to lake species phylogeny and ecology, suggesting GRN rewiring in radiating cichlids. Conclusions Our approach reveals numerous novel potential candidate regulators and regulatory regions across cichlid genomes, including some novel and some previously reported associations to known adaptive evolutionary traits.

Evolution of regulatory networks associated with traits under selection in cichlids

Seminal studies of vertebrate protein evolution speculated that gene regulatory changes can drive anatomical innovations. However, very little is still known about gene regulatory network (GRN) evolution associated with phenotypic effect across ecologically-diverse species. Using a novel approach to reconstruct GRNs in vertebrate species, we aimed to study GRN evolution in representative species of the most striking example of an adaptive radiation, the East African cichlids. We previously demonstrated how the explosive phenotypic diversification of East African cichlids is attributed to diverse molecular mechanisms, including accelerated regulatory sequence evolution and gene expression divergence. To investigate these mechanisms across species at a genome-wide scale, our novel network-based approach identifies ancestral and extant gene co-expression modules along a phylogeny, and by integrating associated regulators, predicts candidate regulatory regions implicated in traits under selection in cichlids. As a case study, we present data from a well-studied adaptive trait - the visual system - for which we report striking cases of network rewiring for visual opsin genes, identify discrete regulatory variants, and investigate the plausibility of their association with cichlid visual system evolution. In regulatory regions of visual opsin genes, in vitro assays confirm that transcription factor binding site mutations disrupt regulatory edges across species, and segregate according to lake species phylogeny and ecology, suggesting GRN rewiring in radiating cichlids. Our approach revealed numerous novel potential candidate regulatory regions across cichlid genomes with no prior association, as well as those with previously reported associations to known adaptive evolutionary traits, thus providing proof of concept.