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.

Fishes use olfaction to gain varied information vital for survival and communication. To understand biodiversity in fishes, it is important to identify what receptors individual fish use to detect specific chemical compounds. However, studies of fish olfactory receptors and their ligands are still limited to a few model organisms represented primarily by zebrafish. Here, we tested the c-fos expression of olfactory sensory neurons (OSNs) in an East African cichlid, the most diversified teleost lineage, by in situ hybridization with a c-fos riboprobe. We confirmed that microvillous neurons contributed the most to the detection of amino acids, as in other fishes. Conversely, we found that ciliated neurons contributed the most to detection of conjugated steroids, known as pheromone candidates. We also found that V2Rs, the major receptor type in microvillous neurons, exhibited differential responsiveness to amino acids, and further suggest that the cichlid-specific duplication of V2R led to ligand differentiation by demonstrating a differential response to arginine. Finally, we established a nonlethal method to collect cichlid urine and showed how various OSNs, including V1R+ neurons, respond to male urine. This study provides an experimental basis for understanding how cichlids encode natural odors, which ultimately lead to provide insight into how olfaction has contributed to the diversification of cichlids.