Shoji Fukamachi’s research while affiliated with Japan Women's University and other places

Individual identification is an important ability for humans and perhaps also for non-human animals to lead social lives. It is also desirable for laboratory experiments to keep records of each animal while rearing them in mass. However, the specific body parts or the acceptable visual angles that enable individual identification are mostly unknown for non-human animals. In this study, we investigated whether artificial intelligence (AI) could distinguish individual medaka, a model animal for biological, agrarian, ecological, and ethological studies, based on the dorsal view. Using Teachable Machine, we took photographs of adult fish (n = 4) and used the images for machine learning. To our surprise, the AI could perfectly identify the four individuals in a total of 11 independent experiments, and the identification was valid for up to 10 days. The AI could also distinguish eight individuals, although machine learning required more time and effort. These results clearly demonstrate that the dorsal appearances of this small spot-/stripe-less fish are polymorphic enough for individual identification. Whether these clues can be applied to laboratory experiments where individual identification would be beneficial is an intriguing theme for future research.

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.

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.

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.

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.

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.

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.

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.