Elsevier

Gene

Volume 499, Issue 2, 15 May 2012, Pages 273-279
Gene

Characterization of V1R receptor (ora) genes in Lake Victoria cichlids

https://doi.org/10.1016/j.gene.2012.03.002Get rights and content

Abstract

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.

Highlights

► We identified all six intact V1R genes (V1R1 to V1R6) in Lake Victoria cichlids. ► All V1R mRNAs are expressed in the olfactory epithelium of Lake Victoria cichlids. ► Lake Victoria cichlids may use V1R-mediated olfaction in their social behavior.

Introduction

Most fish rely on olfactory traits to mediate many kinds of social behaviors including feeding, reproduction, migration, kin recognition, and aggression (Laberge and Hara, 2001, Sorensen and Stacey, 2004). Olfactory cues and the relevant behaviors have been identified in various fishes; these cues include migratory pheromones in sea lamprey (Sorensen et al., 2005), priming pheromones in goldfish (Dulka et al., 1987, Kobayashi et al., 2002), and a male-attracting pheromone in masu salmon (Yambe et al., 2006). Because chemical signaling could be advantageous under water, each fish has developed a highly sophisticated olfactory system for social communication. However, the importance of olfactory communication in species-rich African cichlids remains to be elucidated.

The species flocks of the East African Great Lakes—Lakes Tanganyika, Malawi, and Victoria—comprise several hundred endemic cichlid species that are ecologically and morphologically highly diverse and are textbook examples of adaptive radiation (Fryer and Iles, 1972). Kocher (2004) categorized the cichlid speciation events into three stages: (i) choice of habitat, (ii) morphological diversification and change of trophic status, and (iii) diversification of color pattern and of mate recognition. Thus, visual cues are of primary importance for the last stage of cichlid radiation. Indeed, several molecular studies (Seehausen et al., 2008, Sugawara et al., 2005, Terai et al., 2002, Terai et al., 2006) have shown adaptive and divergent evolution of opsin genes, suggesting the importance of visual cues for cichlid radiation. In contrast, olfaction has received less attention from biologists. Several recent studies, however, proposed that olfaction also contributes substantially to cichlid reproductive communication and mating. For example, Crapon de Caprona (1980) showed that male cichlids native to Lake Tanganyika, Haplochromis burtoni, rapidly change their behavior and color by responding to the odor of females in the reproductive state, suggesting that they partly use reproductive pheromones in this species. Furthermore, a series of electro-olfactogram recording studies demonstrated that H. burtoni can detect a variety of conjugated steroids, which have pheromonal functions in gobies and African cat fish (Cole and Stacey, 2003, Cole and Stacey, 2006, Robison et al., 1998). Detection of putative reproductive pheromones was also demonstrated by Mozambique tilapia (Barata et al., 2008, Miranda et al., 2005). Furthermore, other recent studies showed that mating preference may partly be governed by olfactory cues in cichlids (Plenderleith et al., 2005, Verzijden and ten Cate, 2007). These reports imply that olfaction plays a greater role in cichlid social communication than previously thought.

V1R (also known as ora) is a recently characterized fish olfactory receptor gene family (Pfister and Rodriguez, 2005, Pfister et al., 2007, Saraiva and Korsching, 2007). The repertoire of the fish V1R gene family is small (i.e., six genes in most species), and it is rigidly maintained in five distantly related model fish species, with the exception that fugu and pufferfish lack V1R2 (Saraiva and Korsching, 2007). Furthermore, the sequences of orthologous V1R genes are highly conserved among distantly related rockfish species (Johansson and Banks, 2011) and also among salmonid species (Johnson and Banks, 2011). These attributes are in striking contrast to other olfactory receptor gene families (e.g., OR, V2R, TAAR), which have large and highly variable gene repertoires owing to extensive lineage-specific expansions (Hashiguchi et al., 2008, Hussain et al., 2009, Nei et al., 2008, Niimura and Nei, 2005). Although the functions of fish V1R olfactory receptors are currently unknown, these unique features imply that they can recognize a small set of evolutionarily conserved chemicals, such as reproductive pheromones (Johansson and Banks, 2011, Saraiva and Korsching, 2007). Indeed, the metabolites of the hormones such as prostaglandins or steroids once used for sexual maturation are sometimes diverted to reproductive pheromones which might be involved in assessing mate condition or reproductive status (Dulka et al., 1987). Accordingly, it is worthwhile to investigate the V1R receptors in cichlids to understand the possible involvement of olfaction in their social communication, especially in reproduction. In the present study, we explored the V1R genes of the Lake Victoria cichlid, Haplochromis chilotes, by screening the bacterial artificial chromosome (BAC) library of this species. We found six intact V1R genes, all of which are orthologous to the known V1R genes. Furthermore, by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization analyses, we detected the expression of mRNA for these V1R genes in Haplochromis sauvagei, suggesting they are all functional. This study indicates that the cichlids utilize V1R-mediated olfaction as well as vision in their reproduction.

Section snippets

Fish and DNA samples

Two Lake Victoria cichlids; H. chilotes and H. sauvagei, were used in the present study. The cichlids were caught during explorations that were led by the Okada laboratory from 2005 to 2007. These fish were kept and raised in aquaria in the laboratory. Tissues from fresh-caught fish were fixed in 100% ethanol and stored at 4 °C. DNA was extracted using the DNeasy tissue kit (Qiagen), then preserved at 4 °C.

PCR and DNA sequencing

PCR was carried out with 10–100 ng of total genomic DNA, 1 U of Ex-Taq DNA polymerase (using

Isolation of cichlid V1R genes

We first isolated partial sequences of the V1R genes by PCR using degenerate primer sets, which were designed to target the conserved region of the nucleotide alignment among five teleost fish (medaka, stickleback, fugu, pufferfish, zebrafish). The sequences of these degenerate primers are shown in Table 1A. The total genomic DNA of the Lake Victoria cichlid H. chilotes was used for the initial PCR. Partial sequences of the V1R genes in H. chilotes were successfully obtained for all of the V1R

Acknowledgments

We thank the Tanzania Commission for Science and Technology (COSTECH) for research permission, and the Tanzania Fisheries Research Institute (TAFIRI) for permission to use their facilities. We also thank S. Mizoiri, M. Aibara, M. M. Kayeba, and M. Haruna for collecting cichlids and A. Imai and A. Nakanishi for technical assistance. This work was supported by research grants from JSPS AA Science Platform Program and from the Ministry of Education, Culture, Sports, Science and Technology of Japan

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