Behavioral photosensitivity of multi-color-blind medaka: enhanced response under ultraviolet light in the absence of short-wavelength-sensitive opsins.

BACKGROUND: The behavioral photosensitivity of animals could be quantified via the optomotor response (OMR), for example, and the luminous efficiency function (the range of visible light) should largely rely on the repertoire and expression of light-absorbing proteins in the retina, i.e., the opsins. In fact, the OMR under red light was suppressed in medaka lacking the red (long-wavelength sensitive [LWS]) opsin. RESULTS: We investigated the ultraviolet (UV)- or blue-light sensitivity of medaka lacking the violet (short-wavelength sensitive 1 [SWS1]) and blue (SWS2) opsins. The sws1/sws2 double or sws1/sws2/lws triple mutants were as viable as the wild type. The remaining green (rhodopsin 2 [RH2]) or red opsins were not upregulated. Interestingly, the OMR of the double or triple mutants was equivalent or even increased under UV or blue light (λ = 350, 365, or 450 nm), which demonstrated that the rotating stripes (i.e., changes in luminance) could fully be recognized under UV light using RH2 alone. The OMR test using dichromatic stripes projected onto an RGB display consistently showed that the presence or absence of SWS1 and SWS2 did not affect the equiluminant conditions. CONCLUSIONS: RH2 and LWS, but not SWS1 and SWS2, should predominantly contribute to the postreceptoral processes leading to the OMR or, possibly, to luminance detection in general, as the medium-wavelength-sensitive and LWS cones, but not the SWS cones, are responsible for luminance detection in humans.

An Attempt to Identify the Medaka Receptor for Somatolactin Alpha Using a Reverse Genetics Approach.

Somatolactin alpha (SLα) is a fish-specific hormone involved in body color regulation. The growth hormone (GH) is another hormone that is expressed in all vertebrates and promotes growth. These peptide hormones act by binding to receptors (SLα receptor (SLR) and GH receptor (GHR)); however, the relationships between these ligands and their receptors vary among species. Here, we first performed phylogenetic tree reconstruction by collecting the amino-acid sequences classified as SLR, GHR, or GHR-like from bony fish. Second, we impaired SLR or GHR functions in medaka (Oryzias sakaizumii) using CRISPR/Cas9. Lastly, we analyzed SLR and GHR mutants for phenotypes to deduce their functions. Phylogenetic tree reconstruction was performed using a total of 222 amino-acid sequences from 136 species, which revealed that many GHRa and GHRb are vaguely termed as GHR or GHR-like, while showing no orthologous/paralogous relationships. SLR and GHR mutants were successfully established for phenotyping. SLR mutants exhibited premature lethality after hatching, indicating an essential role for SLR in normal growth. GHR mutations did not affect viability, body length, or body color. These results provide no evidence that either SLR or GHR functions as a receptor for SLα; rather, phylogenetically and functionally, they seem to be receptors for GH, although their (subfunctionalized) roles warrant further investigation.

Retinal Cone Mosaic in sws1-Mutant Medaka (Oryzias latipes), A Teleost.

Purpose: Ablation of short single cones (SSCs) expressing short-wavelength-sensitive opsin (SWS1) is well analyzed in the field of regenerative retinal cells. In contrast with ablation studies, the phenomena caused by the complete deletion of SWS1 are less well-understood. To assess the effects of SWS1 deficiency on retinal structure, we established and analyzed sws1-mutant medaka. Methods: To visualize SWS1, a monoclonal anti-SWS1 antibody and transgenic reporter fish (Tg(sws1:mem-egfp)) were generated. We also developed a CRISPR/Cas-driven sws1-mutant line. Retinal structure of sws1 mutant was visualized using anti-SWS1, 1D4, and ZPR1 antibodies and coumarin derivatives and compared with wild type, Tg(sws1:mem-egfp), and another opsin (lws) mutant. Results: Our rat monoclonal antibody specifically recognized medaka SWS1. Sws1 mutant retained regularly arranged cone mosaic as lws mutant and its SSCs had neither SWS1 nor long wavelength sensitive opsin. Depletion of sws1 did not affect the expression of long wavelength sensitive opsin, and vice versa. ZPR1 antibody recognized arrestin spread throughout double cones and long single cones in wild-type, transgenic, and sws1-mutant lines. Conclusions: Comparative observation of sws1-mutant and wild-type retinas revealed that ZPR1 negativity is not a marker for SSCs with SWS1, but SSCs themselves. Loss of functional sws1 did not cause retinal degeneration, indicating that sws1 is not essential for cone mosaic development in medaka. Our two fish lines, one with visualized SWS1 and the other lacking functional SWS1, offer an opportunity to study neural network synapsing with SSCs and to clarify the role of SWS1 in vision.

Validation of the three-chamber strategy for studying mate choice in medaka.

The three-chamber experiment, in which one test animal can choose between two animals placed in physically inaccessible compartments, is a widely adopted strategy for studying sexual preference in animals. Medaka, a small freshwater teleost, is an emerging model for dissecting the neurological/physiological mechanisms underlying mate choice for which intriguing findings have been accumulating. The three-chamber strategy has rarely been adopted in this species; therefore, here we investigated its validity using medaka colour variants that mate assortatively. First, a total of 551 movies, in which a test male and two choice females interacted for 30 min under a free-swimming condition, were manually analysed. The sexual preference of the males, calculated as a courtship ratio, was highly consistent between human observers (r > 0.96), supporting the objectivity of this manual-counting strategy. Second, we tested two types of three-chamber apparatuses, in which choice fish were presented in either a face-to-face or side-by-side location. Test fish (regardless of sex) spent most of the time associating with choice fish in the compartments. However, their sexual preference, calculated as an association ratio, was poorly reproduced when the locations of the choice fish were swapped. Third, the sexual preferences of males quantified using the manual-counting and either of the three-chamber strategies did not correlate (r = 0.147 or 0.297). Hence, we concluded that, even for individuals of a species like medaka, which spawn every day, sexual preference could not be reliably evaluated using the three-chamber strategy. Optimization of the protocol may solve this problem; however, the explanation for the observation that animals that are ready for spawning persist with never-accessible mating partners must be reconsidered.

Behavioural red-light sensitivity in fish according to the optomotor response.

Various procedures have been adopted to investigate spectral sensitivity of animals, e.g. absorption spectra of visual pigments, electroretinography, optokinetic response, optomotor response (OMR) and phototaxis. The use of these techniques has led to various conclusions about animal vision. However, visual sensitivity should be evaluated consistently for a reliable comparison. In this study, we retrieved behavioural data of several fish species using a single OMR procedure and compared their sensitivities to near-infrared light. Besides cavefish that lack eyes, some species were not appropriate for the OMR test because they either stayed still or changed swimming direction frequently. Eight of 13 fish species tested were OMR positive. Detailed analyses using medaka, goldfish, zebrafish, guppy, stickleback and cichlid revealed that all the fish were sensitive to light at a wavelength greater than or equal to 750 nm, where the threshold wavelengths varied from 750 to 880 nm. Fish opsin repertoire affected the perception of red light. By contrast, the copy number of long-wavelength-sensitive (LWS) genes did not necessarily improve red-light sensitivity. While the duplication of LWS and other cone opsin genes that has occurred extensively during fish evolution might not aid increasing spectral sensitivity, it may provide some other advantageous ophthalmic function, such as enhanced spectral discrimination.

Changes in a Cone Opsin Repertoire Affect Color-Dependent Social Behavior in Medaka but Not Behavioral Photosensitivity.

Common ancestors of vertebrates had four types of cone opsins: short-wavelength sensitive 1 (SWS1), SWS2, rhodopsin 2 (RH2), and long-wavelength sensitive (LWS) types. Whereas fish and birds retain all the types, mammals have lost two of them (SWS2 and RH2) possibly because of their nocturnal lifestyle during the Mesozoic Era. Considering that the loss of cone opsin types causes so-called color blindness in humans (e.g., protanopia), the ability to discriminate color by trichromatic humans could be lower than that in potentially tetrachromatic birds and fish. Behavioral studies using color-blind (cone opsin-knockout) animals would be helpful to address such questions, but it is only recently that the genome-editing technologies have opened up this pathway. Using medaka as a model, we introduced frameshift mutations in SWS2 (SWS2a and/or SWS2b) after detailed characterization of the loci in silico, which unveiled the existence of a GC-AG intron and non-optic expressed-sequence-tags (ESTs) that include SWS2a in part. Transcripts from the mutated SWS2 loci are commonly reduced, suggesting that the SWS2a/b-double mutants could produce, if any, severely truncated (likely dysfunctional) SWS2s in small amounts. The mutants exhibited weakened body color preferences during mate choice. However, the optomotor response (OMR) test under monochromatic light revealed that the mutants had no defect in spectral sensitivity, even at the absorbance maxima (λmax) of SWS2s. Evolutionary diversification of cone opsins has often been discussed in relation to adaptation to dominating light in habitats (i.e., changes in the repertoire or λmax are for increasing sensitivity to the dominating light). However, the present results seem to provide empirical evidence showing that acquiring or losing a type of cone opsin (or changes in λmax) need not substantially affect photopic or mesopic sensitivity. Other points of view, such as color discrimination of species-specific mates/preys/predators against habitat-specific backgrounds, may be necessary to understand why cone opsin repertories are so various among animals.

Evolutionary history of the medaka long-wavelength sensitive genes and effects of artificial regression by gene loss on behavioural photosensitivity.

Tandem gene duplication has led to an expansion of cone-opsin repertoires in many fish, but the resulting functional advantages have only been conjectured without empirical demonstration. Medaka (Oryzias latipes and O. sakaizumii) have eight (two red, three green, two blue, and one violet) cone opsin genes. Absorbance maxima (λmax) of the proteins vary from 356 nm to 562 nm, but those of the red opsins (long-wavelength sensitive; LWS) are nearly identical, obscuring the necessity of their coexistence. Here, we compared the LWSa and LWSb loci of these sister species and found that the gene duplication occurred long before the latipes-sakaizumii speciation (4-18 million years ago), and the high sequence similarity between the paralogues is the result of at least two events of gene conversion. These repetitive gene conversions would indicate the importance for medaka of retaining two identical LWSs in the genome. However, a newly established medaka mutant with a single LWS showed no defect in LWS expression or behavioural red-light sensitivity, demonstrating functional redundancy of the paralogs. Thus, as with many other genes after whole-genome duplication, the redundant LWS might be on the way to being lost from the current cone opsin repertoire. Thus, non-allelic gene conversion may temporarily provide an easier and more frequent solution than gene loss for reducing genetic diversity, which should be considered when assessing history of gene evolution by phylogenetic analyses.

A semi-automatic and quantitative method to evaluate behavioral photosensitivity in animals based on the optomotor response (OMR).

The optomotor response (OMR) is a locomotor behavior of animals that is induced by moving repetitive visual stimuli. This characteristic helps animals particularly when stabilizing and maintaining position in schools and herds. Here, we developed a simple but sensitive method for quantifying the OMR using medaka (Oryzias latipes) as a model. This method, which simply requires video-recorded behavior, free tracking software, and a generic spreadsheet program, enables the evaluation of spectral sensitivity by researchers with little knowledge about the behavioral characteristics of the test animal or of the OMR. Based on a manual method, we reported previously that wild-type and red-colorblind medaka exhibited an OMR up to λ=830 and 740 nm, respectively. However, the present method, which quantifies the OMR according to three parameters (starting time, duration, and total distance of swimming) that are calculated based on a series of x-y coordinates of the moving fish, supported that conclusion and further indicated that both strains perceive light at even longer wavelengths. This low-cost, quantitative, and semi-automatic method would widen the opportunities to unveil behavioral photosensitivity in animals of interest.This article has an associated First Person interview with the first author of the paper.

Loss of red opsin genes relaxes sexual isolation between skin-colour variants of medaka.

Colour vision is often essential for animals. Fine discrimination of colours enhances the ability of animals to find food, predators, or mating partners. Using two colour variants of medaka (Oryzias latipes), which mate assortatively depending on visual cues (pale grey versus dark orange), we recently established red colour-blind strains by knocking out the red opsin (long-wavelength-sensitive) genes and elucidated that the fish were indeed insensitive to red light. In the present study, we investigated the mate choice of these red-blind fish. The colour variants with normal colour vision strongly preferred to mate with their own strain. The red-blind ones also preferred their own strain; i.e. they still mated assortatively. However, their preference was significantly weaker than that of fish with normal colour vision. In other words, the red-blind fish showed increased sexual interest in the other colour variant. These results indicated that reduced sensitivity to red light also reduced their ability to discriminate colours. This empirical evidence directly demonstrates that a change in cone-opsin repertoire changes mating decision behaviours, which would affect gene flow and speciation processes between conspecific colour variants in nature, as suggested in other studies.

Dynamic plasticity in phototransduction regulates seasonal changes in color perception.

To cope with seasonal changes in the environment, organisms adapt their physiology and behavior. Although color perception varies among seasons, the underlying molecular basis and its physiological significance remain unclear. Here we show that dynamic plasticity in phototransduction regulates seasonal changes in color perception in medaka fish. Medaka are active and exhibit clear phototaxis in conditions simulating summer, but remain at the bottom of the tank and fail to exhibit phototaxis in conditions simulating winter. Mate preference tests using virtual fish created with computer graphics demonstrate that medaka are more attracted to orange-red-colored model fish in summer than in winter. Transcriptome analysis of the eye reveals dynamic seasonal changes in the expression of genes encoding photopigments and their downstream pathways. Behavioral analysis of photopigment-null fish shows significant differences from wild type, suggesting that plasticity in color perception is crucial for the emergence of seasonally regulated behaviors.Animal coloration and behavior can change seasonally, but it is unclear if visual sensitivity to color shifts as well. Here, Shimmura et al. show that medaka undergo seasonal behavioral change accompanied by altered expression of opsin genes, resulting in reduced visual sensitivity to mates during winter-like conditions.

Establishment and maintenance of sexual preferences that cause a reproductive isolation between medaka strains in close association.

Animals choose reproductive partners based on their sexual preferences which are established at a certain time point before, during, or after sexual maturation. The preferences are often divergent within a species, which suppresses gene flow between populations and may promote speciation. There are two strains of medaka (Oryzias latipes) that differ by a single transgene and mate assortatively depending on skin color. Here, we demonstrate that symmetrically biased (mutually exclusive) sexual preferences are (1) gradually established during growth depending on skin color and the color of surrounding fish, (2) strong enough to minimize gene flow between the strains at a population level, and (3) inflexibly retained after sexual maturation, even after weeks of daily mating with partners of the other strain. Thus, these laboratory strains of medaka are under premating isolation with the simplest genomic structure. They provide an empirical platform for assessing the complex and hypothetical mechanisms of speciation by mate choice.

Protanopia (red color-blindness) in medaka: a simple system for producing color-blind fish and testing their spectral sensitivity.

BACKGROUND: Color perception is important for fish to survive and reproduce in nature. Visual pigments in the retinal photoreceptor cells are responsible for receiving light stimuli, but the function of the pigments in vivo has not been directly investigated in many animals due to the lack of color-blind lines and appropriate color-perception tests. METHODS: In this study, we established a system for producing color-blind fish and testing their spectral sensitivity. First, we disrupted long-wavelength-sensitive (LWS) opsins of medaka (Oryzias latipes) using the CRISPR/Cas9 system to make red-color-blind lines. Single guide RNAs were designed using the consensus sequences between the paralogous LWSa and LWSb genes to simultaneously introduce double-frameshift mutations. Next, we developed a non-invasive and no-prior-learning test for spectral sensitivity by applying an optomotor response (OMR) test under an Okazaki Large Spectrograph (OLS), termed the O-O test. We constructed an electrical-rotary cylinder with black/white stripes, into which a glass aquarium containing one or more fish was placed under various monochromatic light conditions. The medaka were irradiated by the OLS every 10 nm, from wavelengths of 700 nm to 900 nm, and OMR was evaluated under each condition. RESULTS: We confirmed that the lws - medaka were indeed insensitive to red light (protanopia). While the control fish responded to wavelengths of up to 830 nm (λ = 830 nm), the lws - mutants responded up to λ = 740 nm; however, this difference was not observed after adaptation to dark: both the control and lws - fish could respond up to λ = 820 ~ 830 nm. CONCLUSIONS: These results suggest that the lws - mutants lost photopic red-cone vision, but retained scotopic rod vision. Considering that the peak absorption spectra (λmax) of medaka LWSs are about 560 nm, but the light-adapted control medaka could respond behaviorally to light at λ = 830 nm, red-cone vision could cover an unexpectedly wide range of wavelengths, and behavioral tests could be an effective way to measure spectral sensitivity. Using the CRISPR/Cas9 and O-O systems, the establishment of various other color-blind lines and assessment of their spectra sensitivity could be expected to proceed in the future.

Characterization of assortative mating in medaka: Mate discrimination cues and factors that bias sexual preference.

Somatolactin alpha (SLα) is a peptide hormone that regulates skin color, and SLα-deficient and SLα-excess strains have been established in medaka (Oryzias latipes). Their skin colors differ conspicuously and males prefer to mate with females from the same strain. Pre-mating sexual isolation in this model vertebrate provides an ideal platform for investigating the molecular mechanisms of mate choice. Thus, we studied the sensory cues utilized by these fish to discriminate the same and different strains. When males were given a choice under monochromatic light, where the skin colors differed only in terms of brightness but not in hue, mating occurred but it was not assortative. This suggests that: (1) a visual cue is essential for mate discrimination rather than odor or acoustic cues; (2) the visual cue is color and not shape, size, or motion; and (3) the color cue needs to be perceived as the relative balance of brightness at multiple wavelengths rather than the brightness at a specific wavelength. In addition, we introduced another skin-color mutation into the SLα-excess strain and found that this new strain and the original SLα-excess strain, which also overexpressed SLα but exhibited distinct skin colors, preferred different colors. This demonstrates that SLα is not a primary determinant of sexual preference. The symmetrically biased sexual preferences of the SLα-deficient and SLα-excess strains may be acquired postnatally depending on their individual skin color or that of tank mates.

Transgenic medaka that overexpress growth hormone have a skin color that does not indicate the activation or inhibition of somatolactin-α signal.

Teleosts have two paralogous growth-hormone receptors (GHRs). In vitro studies demonstrated that both receptors bind to and transmit the signal of the growth hormone (GH). However, one of the GHRs (GHR1) was shown to bind more strongly to somatolactin-α (SLα), a fish-specific peptide hormone that is closely related to GH, and is, therefore, termed somatolactin receptor (SLR). In this study, we questioned whether the dual binding of GHR1/SLR causes a crosstalk (reciprocal activation or inhibition) between GH and SLα signals in vivo. For this purpose, we newly established a transgenic medaka that overexpresses GH (Actb-GH:GFP) and assessed its phenotype. The body weight of these transgenic medaka is about twice that of wild-type fish, showing that functional GH was successfully overexpressed in Actb-GH:GFP fish. The transgenic medaka, especially female fish, showed severe infertility, which was a common side effect in GH transgenesis. The skin color, which reflects the effects of SLα most conspicuously in medaka, was similar to that of neither the SLα-overexpressing nor the SLα-deficient medaka, indicating that GH overexpression does not enhance or suppress the SLα signal. We also verified that a transgenic medaka that overexpressed SLα grew and reproduced normally. Therefore, regardless of the in vitro binding relationships, the GH and SLα signals seem not to crosstalk significantly in vivo even when these hormones are overexpressed.

Effects of body-color mutations on vitality: an attempt to establish easy-to-breed see-through medaka strains by outcrossing.

"See-through" strains of medaka are unique tools for experiments: their skin is transparent, and their internal organs can be externally monitored throughout life. However, see-through fish are less vital than normally pigmented wild-type fish, which allows only skilled researchers to make the most of their advantages. Expecting that hybrid vigor (heterosis) would increase the vitality, we outcrossed two see-through strains (SK(2) and STIII) with a genetically distant wild-type strain (HNI). Fish with the see-through phenotypes were successfully restored in the F2 generation and maintained as closed colonies. We verified that genomes of these hybrid see-through strains actually consisted of approximately 50% HNI and approximately 50% SK(2) or STIII alleles, but we could not obtain evidence supporting improved survival of larvae or fecundity of adults, at least under our breeding conditions. We also found that four of the five see-through mutations (b(g8), i-3, gu, and il-1 but not lf) additively decrease viability. Given that heterosis could not overwhelm the viability-reducing effects of the see-through mutations, easy-to-breed see-through strains will only be established by other methods such as conditional gene targeting or screening of new body-color mutations that do not reduce viability.

Establishment of HRAS(G12V) transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs.

Most targeted anticancer drugs have been identified by screening at the molecular or cellular level in vitro. However, many compounds selected by such costly and time-consuming screening do not prove effective against tumors in vivo. The development of anticancer drugs would thus be facilitated by the availability of an in vivo screening system based on a multicellular organism. We have now established a transgenic line of the freshwater fish medaka in which melanophores (melanocytes) proliferate in a manner dependent on heat shock-induced signaling by a human RAS oncoprotein. The human HRAS(G12V) oncogene was expressed under the control of a melanophore-specific gene promoter in order to allow visualization of tumor growth in live fish maintained in a water tank. The expression of HRAS(G12V) was induced as a result of Cre-mediated recombination by exposure of the fish to a temperature of 37°C for 30 min, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. The transgenic medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.

Reassessment of the function of somatolactin alpha in lipid metabolism using medaka mutant and transgenic strains.

BACKGROUND: Somatolactin alpha (SLa) is a fish-specific peptide hormone secreted from the pituitary. In medaka, SLa functions to darken the skin color and lack of SLa makes it pale. Transcription of SLa is enhanced or suppressed when fish are kept in dark or bright conditions, respectively, indicating SLa's important role in background acclimation of the skin color. Bizarrely, however, the lack of SLa seems to cause the additional defect of increased triglycerides in organs, which could not be rescued (decreased) by its overexpression. RESULTS: To assess this enigmatic result, we investigated genetic (the SLa, Slc45a2, r, and Y genes) and nongenetic (age, fasting, water temperature, and background color) effects on hepatic triglycerides. These experiments found that percent hepatic triglycerides quickly change in response to external/internal environments. Effects of SLa seemed to be much less obvious, although it may increase the proportion of hepatic triglycerides at least during certain breeding conditions or under certain genetic backgrounds. CONCLUSIONS: The present results do not exclude the possibility that SLa takes part in lipid metabolism or other physiological processes. However, we suggest that skin-color regulation is the only definite role of SLa so far demonstrated in this species.

Genetic, comparative genomic, and expression analyses of the Mc1r locus in the polychromatic Midas cichlid fish (Teleostei, Cichlidae Amphilophus sp.) species group.

Natural populations of the Midas cichlid species in several different crater lakes in Nicaragua exhibit a conspicuous color polymorphism. Most individuals are dark and the remaining have a gold coloration. The color morphs mate assortatively and sympatric population differentiation has been shown based on neutral molecular data. We investigated the color polymorphism using segregation analysis and a candidate gene approach. The segregation patterns observed in a mapping cross between a gold and a dark individual were consistent with a single dominant gene as a cause of the gold phenotype. This suggests that a simple genetic architecture underlies some of the speciation events in the Midas cichlids. We compared the expression levels of several candidate color genes Mc1r, Ednrb1, Slc45a2, and Tfap1a between the color morphs. Mc1r was found to be up regulated in the gold morph. Given its widespread association in color evolution and role on melanin synthesis, the Mc1r locus was further investigated using sequences derived from a genomic library. Comparative analysis revealed conserved synteny in relation to the majority of teleosts and highlighted several previously unidentified conserved non-coding elements (CNEs) in the upstream and downstream regions in the vicinity of Mc1r. The identification of the CNEs regions allowed the comparison of sequences from gold and dark specimens of natural populations. No polymorphisms were found between in the population sample and Mc1r showed no linkage to the gold phenotype in the mapping cross, demonstrating that it is not causally related to the color polymorphism in the Midas cichlid.

Dual control by a single gene of secondary sexual characters and mating preferences in medaka.

BACKGROUND: Animals utilize a wide variety of tactics to attract reproductive partners. Behavioral experiments often indicate an important role for visual cues in fish, but their molecular basis remains almost entirely unknown. Studies on model species (such as zebrafish and medaka) allow investigations into this fundamental question in behavioral and evolutionary biology. RESULTS: Through mate-choice experiences using several laboratory strains of various body colors, we successfully identified one medaka mutant (color interfere; ci) that is distinctly unattractive to reproductive partners. This unattractiveness seems to be due to reduced orange pigment cells (xanthophores) in the skin. The ci strain carries a mutation on the somatolactin alpha (SLa) gene, therefore we expected over-expression of SLa to make medaka hyper-attractive. Indeed, extremely strong mating preferences were detected in a choice between the ci and SLa-transgenic (Actb-SLa:GFP) medaka. Intriguingly, however, the strains showed opposite biases; that is, the mutant and transgenic medaka liked to mate with partners from their own strain, similar to becoming sexually isolated. CONCLUSION: This study spotlighted SLa as a novel mate-choice gene in fish. In addition, these results are the first demonstration of a single gene that can pleiotropically and harmoniously change both secondary sexual characters and mating preferences. Although theoretical models have long suggested joint evolution of linked genes on a chromosome, a mutation on a gene-regulatory region (that is, switching on/off of a single gene) might be sufficient to trigger two 'runaway' processes in different directions to promote (sympatric) speciation.

Effects of constitutive expression of somatolactin alpha on skin pigmentation in medaka.

Somatolactin alpha (SLa) is a fish-specific peptide hormone secreted from the pituitary. Its functions have been investigated for the last decade but are still under debate. We previously reported a frame-shift mutation on SLa in a medaka mutant, color interfere (ci), which shows defects in body-color regulation, lipid storage, and cortisol secretion. In this study, we examined the effects of introducing a DNA fragment which constitutively expresses wild-type SLa under regulation of the beta-actin (Actb) promoter into the ci genome. We successfully visualized the transgene expression by taking advantage of an internal ribosome entry site (IRES) and the green fluorescent protein (GFP). The transgenic medaka, Actb-SLa:GFP, exhibited a reversed body-color phenotype of ci; more orange xanthophores and less white leucophores. We also detected more black melanophores and less silver iridophores, which indicates SLa's comprehensive role in regulating all types of pigment cells in the skin. Unexpectedly, the defects in lipid/cortisol contents remained in Actb-SLa:GFP. Therefore, the causal relationship between SLa and lipid/cortisol metabolisms relapses to an open question which needs to be reassessed by other types of experiments. Both the Actb-SLa:GFP and ci fish grow and mature similar to wild type, indicating SLa contributes little to growth regulation in spite of the fact that it binds to a teleost-specific paralog of growth-hormone receptor in vitro. The present study provides definitive evidence for SLa's principal and indispensable role in body-color regulation in medaka.