Dynamics of Spermatogenesis and Change in Testicular Morphology under 'Mating' and 'Non-Mating' Conditions in Medaka (Oryzias latipes).

Here, we report that the gross morphology of the testes changes under 'non-mating' or 'mating' conditions in medaka (Oryzias latipes). During these conditions, an efferent duct expands and a histological unit of spermatogenesis, the lobule, increases its number under 'non-mating' conditions. Based on BrdU labeling experiments, lower mitotic activity occurs in gonial cells under 'non-mating' conditions, which is consistent with the reduced number of germ cell cysts. Interestingly, the total number of type A spermatogonia was maintained, regardless of the mating conditions. In addition, the transition from mitosis to meiosis may have been retarded under the 'non-mating' conditions. The minimum time required for germ cells to become sperm, from the onset of commitment to spermatogenesis, was approximately 14 days in vivo. The time was not found to significantly differ between 'non-mating' and 'mating' conditions. The collective data suggest the presence of a mechanism wherein the homeostasis of spermatogenesis is altered in response to the mating conditions.

3D Atlas of the Pituitary Gland of the Model Fish Medaka (Oryzias latipes).

In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their physiological functions. Using medaka as a model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba-, pomca-, and lhb-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its functional plasticity.

Both of marine fish species Oryzias melastigma and Pagrus major all failing in early localization at embryo stage by vasa RNA.

Germ cells are essential for gonadal development. As precursors of germ cells, primordial germ cells (PGCs) are particularly important for germline formation. However, the research on distribution patterns of PGCs in marine fish is very limited, especially for economic species. The vasa gene has been widely used as marker to identify PGCs origination and migration because of vasa RNA is a component of germ plasm. In this study, we isolated full-length vasa cDNA (Omvas and Pmvas) from marine medaka (Oryzias melastigma) and red seabream (Pagrus major), detected vasa transcripts in different tissues by RT-PCR and described vasa expression patterns during embryogenesis and gametogenesis by in situ hybridization. At the same time, we also explored the relationship between early distribution of germ plasm components and species evolution. The results demonstrated that deduced amino acid sequence of Omvas and Pmvas shared several conserved motifs of Vasa homologues and high identity with other teleost, and vasa transcripts were exclusively detected in early germ cells of gonad. During embryogenesis, vasa RNA of both fishes, like medaka (Oryzias latipes), failed to localize at cleavage furrows and distributed uniformly throughout each blastomere. This study firstly discovered that the marine economic fish, red seabream, lost vasa RNA early specific localization at cleavage furrows and distinctive distribution in germ cells. In addition, compared with other teleost, we found that early distribution of germ plasm might not relate to species evolution. This will improve our understanding of vasa localization modes in teleost, and facilitate fish germ cell manipulation.

A comparison of the structure, composition and mechanical properties of anosteocytic vertebrae of medaka (O. latipes) and osteocytic vertebrae of zebrafish (D. rerio).

Medaka (O. latipes) and zebrafish (D. rerio) are two teleost fish increasingly used as models to study human skeletal diseases. Although they are similar in size, swimming pattern and many other characteristics, these two species are very distant from an evolutionary point of view (by at least 100 million years). A prominent difference between the skeletons of medaka and zebrafish is the total absence of osteocytes in medaka (anosteocytic), while zebrafish bone contains numerous osteocytes (osteocytic). This fundamental difference suggests the possibility that the bony elements of their skeleton may be different in a variety of other aspects, structural, mechanical or both, particularly in heavily loaded bones like the vertebrae. Here we report on the results of a comparative study that aimed to determine the similarities and differences in medaka and zebrafish vertebrae in terms of their macro- to nanostructure, composition and mechanical properties. Our results reveal many similarities between medaka and zebrafish vertebrae, making the lack or presence of osteocytes the only major difference between the bones of these two species.

Perspectives on sustainable management of the Poso Lake (Indonesia) endemic ricefish, Oryzias nigrimas (Actinopterygii: Adrianichthyidae) / Perspectivas sobre el manejo sostenible del pez Oryzias nigrimas, endémico del Lago Poso en Indonesia (Actinopterygii: Adrianichthyidae)

Introduction: The endemic fishes of the ancient lakes of Sulawesi are under increasing threat. Objective: To evaluate the data and information available from a holistic management perspective and to formulate measures to conserve the endemic ricefish Oryzias nigrimas in Poso Lake, Indonesia. Methods: Collection of primary data from three stations around Lake Poso and literature study. Results: Threats to O. nigrimas include habitat degradation and loss, introduced alien species, and exploitation as a locally important food fish. Options to promote sustainable fisheries management include spatial and temporal limitations to minimise catch of gravid or brooding fish. Habitat protection should include measures to minimise impacts from activities which can reduce water quality and disturb or kill aquatic vegetation. Conclusion: Measures to prevent further O. nigrimas population decline are considered urgent and further research is recommended to fill identified knowledge gaps. Ex-situ conservation, including the development of captive breeding, could also contribute to a holistic O. nigrimas conservation strategy.

Introducción: Los peces endémicos de los antiguos lagos de Sulawesi están cada vez más amenazados. Objetivo: Evaluar los datos y la información disponible desde una perspectiva de gestión holística y formular medidas para conservar el pez endémico Oryzias nigrimas en el lago Poso, Indonesia. Métodos: Recopilación de datos primarios de tres estaciones alrededor del lago Poso y revisión de la literatura. Resultados: Las amenazas para O. nigrimas incluyen la degradación y pérdida del hábitat, la introducción de especies exóticas y la explotación como producto pesquero de importancia local. Las opciones para promover la ordenación pesquera sostenible incluyen limitaciones espaciales y temporales para minimizar la captura de peces grávidos o reproductores. La protección del hábitat debe incluir medidas para minimizar los impactos de las actividades que pueden reducir la calidad del agua y perturbar o matar la vegetación acuática. Conclusión: Las medidas para prevenir una disminución de la población de O. nigrimas se consideran urgentes y se recomienda realizar más investigaciones para llenar los vacíos de conocimiento identificados. La conservación ex situ, incluido el desarrollo de la cría en cautividad, también podría contribuir a una estrategia de conservación holística de O. nigrimas.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes).

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Intact in vivo visualization of telencephalic microvasculature in medaka using optical coherence tomography.

To date, various human disease models in small fish-such as medaka (Oryzias lapties)-have been developed for medical and pharmacological studies. Although genetic and environmental homogeneities exist, disease progressions can show large individual differences in animal models. In this study, we established an intact in vivo angiographic approach and explored vascular networks in the telencephalon of wild-type adult medaka using the spectral-domain optical coherence tomography. Our approach, which required neither surgical operations nor labeling agents, allowed to visualize blood vessels in medaka telencephala as small as about 8 µm, that is, almost the size of the blood cells of medaka. Besides, we could show the three-dimensional microvascular distribution in the medaka telencephalon. Therefore, the intact in vivo imaging via optical coherence tomography can be used to perform follow-up studies on cerebrovascular alterations in metabolic syndrome and their associations with neurodegenerative disease models in medaka.

Multi-template matching: a versatile tool for object-localization in microscopy images.

BACKGROUND: The localization of objects of interest is a key initial step in most image analysis workflows. For biomedical image data, classical image-segmentation methods like thresholding or edge detection are typically used. While those methods perform well for labelled objects, they are reaching a limit when samples are poorly contrasted with the background, or when only parts of larger structures should be detected. Furthermore, the development of such pipelines requires substantial engineering of analysis workflows and often results in case-specific solutions. Therefore, we propose a new straightforward and generic approach for object-localization by template matching that utilizes multiple template images to improve the detection capacity. RESULTS: We provide a new implementation of template matching that offers higher detection capacity than single template approach, by enabling the detection of multiple template images. To provide an easy-to-use method for the automatic localization of objects of interest in microscopy images, we implemented multi-template matching as a Fiji plugin, a KNIME workflow and a python package. We demonstrate its application for the localization of entire, partial and multiple biological objects in zebrafish and medaka high-content screening datasets. The Fiji plugin can be installed by activating the Multi-Template-Matching and IJ-OpenCV update sites. The KNIME workflow is available on nodepit and KNIME Hub. Source codes and documentations are available on GitHub (https://github.com/multi-template-matching). CONCLUSION: The novel multi-template matching is a simple yet powerful object-localization algorithm, that requires no data-pre-processing or annotation. Our implementation can be used out-of-the-box by non-expert users for any type of 2D-image. It is compatible with a large variety of applications including, for instance, analysis of large-scale datasets originating from automated microscopy, detection and tracking of objects in time-lapse assays, or as a general image-analysis step in any custom processing pipelines. Using different templates corresponding to distinct object categories, the tool can also be used for classification of the detected regions.

Machine Learning Methods for Automated Quantification of Ventricular Dimensions.

Medaka (Oryzias latipes) and zebrafish (Danio rerio) contribute substantially to our understanding of the genetic and molecular etiology of human cardiovascular diseases. In this context, the quantification of important cardiac functional parameters is fundamental. We have developed a framework that segments the ventricle of a medaka hatchling from image sequences and subsequently quantifies ventricular dimensions.

Developmental and Full-Life Cycle Exposures to Guanylurea and Guanylurea-Metformin Mixtures Results in Adverse Effects on Japanese Medaka (Oryzias latipes).

Metformin is currently thought to be the highest drug by weight released into the aquatic environment, as a direct result of its widespread use in the treatment of a number of human health disorders. The removal of metformin from wastewaters is directly related to the formation of guanylurea (metformin's only known persistent degradation product), which is generally present at higher concentrations in surface waters than the parent compound. With metformin use rising steadily, it is important to characterize the effects of guanylurea on nontarget aquatic organisms. We recently demonstrated the effects of developmental exposure to environmentally relevant concentrations of metformin on the growth of early life stage (ELS) medaka as well as effects on the body weight of adult male fish following full-life cycle exposures. In the present study, we describe similar effects of guanylurea exposure on these endpoints and life stages. Guanylurea led to effects on growth in a 28-d ELS assessment that were similar to those of metformin; however, these effects occurred at concentrations in the ng/L range compared with the µg/L range for metformin. A possible sex-dependent association with body weight changes was also observed in adults following a 165-d full-life cycle exposure to guanylurea alone or in a mixture with metformin. To our knowledge, the present is the first study to report the toxicity of guanylurea to nontarget aquatic organisms. Environ Toxicol Chem 2019;00:1-6. © 2019 SETAC.

Quantitative morphometric analysis of adult teleost fish by X-ray computed tomography.

Vertebrate models provide indispensable paradigms to study development and disease. Their analysis requires a quantitative morphometric study of the body, organs and tissues. This is often impeded by pigmentation and sample size. X-ray micro-computed tomography (micro-CT) allows high-resolution volumetric tissue analysis, largely independent of sample size and transparency to visual light. Importantly, micro-CT data are inherently quantitative. We report a complete pipeline of high-throughput 3D data acquisition and image analysis, including tissue preparation and contrast enhancement for micro-CT imaging down to cellular resolution, automated data processing and organ or tissue segmentation that is applicable to comparative 3D morphometrics of small vertebrates. Applied to medaka fish, we first create an annotated anatomical atlas of the entire body, including inner organs as a quantitative morphological description of an adult individual. This atlas serves as a reference model for comparative studies. Using isogenic medaka strains we show that comparative 3D morphometrics of individuals permits identification of quantitative strain-specific traits. Thus, our pipeline enables high resolution morphological analysis as a basis for genotype-phenotype association studies of complex genetic traits in vertebrates.

Attraction of posture and motion-trajectory elements of conspecific biological motion in medaka fish.

Visual recognition of conspecifics is necessary for a wide range of social behaviours in many animals. Medaka (Japanese rice fish), a commonly used model organism, are known to be attracted by the biological motion of conspecifics. However, biological motion is a composite of both body-shape motion and entire-field motion trajectory (i.e., posture or motion-trajectory elements, respectively), and it has not been revealed which element mediates the attractiveness. Here, we show that either posture or motion-trajectory elements alone can attract medaka. We decomposed biological motion of the medaka into the two elements and synthesized visual stimuli that contain both, either, or none of the two elements. We found that medaka were attracted by visual stimuli that contain at least one of the two elements. In the context of other known static visual information regarding the medaka, the potential multiplicity of information regarding conspecific recognition has further accumulated. Our strategy of decomposing biological motion into these partial elements is applicable to other animals, and further studies using this technique will enhance the basic understanding of visual recognition of conspecifics.

A rapid and nondestructive protocol for whole-mount bone staining of small fish and Xenopus.

Here we propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. In this procedure, we used a newly developed fixative containing formalin, Triton X-100, and potassium hydroxide, which allows the fixation, decolorization, and transparentization of specimens at the same time. A bone staining solution containing alizarin red S with ethylene glycol and a clearing solution containing Tween 20 and potassium hydroxide also contributed the specificity and swiftness of this new system. As expected, although details of the skeletal system could be observed in specimens with high transparency, it was noteworthy that high-resolution fluorescence images acquired using zoom microscopes clearly delineated the shape of each bone. This new procedure would be expected to be widely used as a standard procedure for bone staining in the testing the developmental toxicity of chemicals and in the screening test of knockout or mutant animals.

Phylogenomics reveals habitat-associated body shape divergence in Oryzias woworae species group (Teleostei: Adrianichthyidae).

The Oryzias woworae species group, composed of O. asinua, O. wolasi, and O. woworae, is widely distributed in southeastern Sulawesi, an island in the Indo-Australian Archipelago. Deep-elongated body shape divergence is evident among these three species to the extent that it is used as a species-diagnostic character. These fishes inhabit a variety of habitats, ranging from upper streams to ponds, suggesting that the body shape divergence among the three species may reflect adaptation to local environments. First, our geometric morphometrics among eight local populations of this species group revealed that the three species cannot be separated by body shape and that riverine populations had more elongated bodies and longer caudal parts than lacustrine populations. Second, their phylogenetic relationships did not support the presence of three species; phylogenies using mitochondrial DNA and genomic data obtained from RNA-Seq revealed that the eight populations could not be sorted into three different clades representing three described species. Third, phylogenetic corrections of body shape variations and ancestral state reconstruction of body shapes demonstrated that body shape divergence between riverine and lacustrine populations persisted even if the phylogenies were considered and that body shape evolved rapidly irrespective of phylogeny. Sexual dimorphism in body shape was also evident, but the degree of dimorphism did not significantly differ between riverine and lacustrine populations after phylogenetic corrections, suggesting that sexual selection may not substantially contribute to geographical variations in body shape. Overall, these results indicate that the deep-elongated body shape divergence of the O. woworae species group evolved locally in response to habitat environments, such as water currents, and that a thorough taxonomic reexamination of the O. woworae species group may be necessary.

Gills of the medaka (Oryzias latipes): A scanning electron microscopy study.

The general morphology and surface ultrastructure of the gills of adult and larvae medaka (Oryzias latipes) were studied in freshwater and seawater using scanning electron microscopy. The gills of all examined fish were structurally similar to those of other teleosts and consisted of four pairs of arches supporting (i) filaments bearing lamellae and (ii) rakers containing taste buds. Three cell types, specifically pavement cells, mitochondria-rich cells (MRCs), and mucous cells, constituted the surface layer of the gill epithelium. Several distinctive characteristics of medaka gills were noted, including the presence of regularly distributed outgrowth on the lamellae, enlarged filament tips, the absence of microridges in most pavement cells in the filament and lamellae and the presence of MRCs in the arch at the filament base. A rapid mode of development was recorded in the gills of larval fish. At hatching, the larvae already had four arches with rudimentary filaments, rakers, and taste buds. The rudimentary lamellae appeared within 2 days after hatching. These results suggest the early involvement of larval gills in respiratory and osmoregulation activities. The responses of the macrostructures and microstructures of gills to seawater acclimation were similar in larvae and adult fish and included modification of the apical surface of MRCs, confirming the importance of these cells in osmoregulation. The potential roles of these peculiarities of the macrostructures and microstructures of medaka gills in the major functions of this organ, such as respiration and osmoregulation, are discussed.

An eye on light-sheet microscopy.

This chapter introduces the principles and advantages of selective plane illumination microscopy (SPIM) and compares it to commonly used epifluorescence or confocal setups. Due to the low phototoxicity, speed of imaging, high penetration depth, and spatiotemporal resolution, SPIM is predestined for in vivo imaging but can as well be used for in toto analysis of large fixed samples. Key points of light-sheet microscopy are highlighted and discussed priming the investigator to choose the best suitable system from the large collection of possible SPIM setups. Mounting of samples is shown and the demands for data acquisition, processing, handling, and visualization are discussed.

Anatomical features for the adequate choice of experimental animal models in biomedicine: I. Fishes.

Fish constitute the oldest and most diverse class of vertebrates, and are widely used in basic research due to a number of advantages (e.g., rapid development ex-utero, large-scale genetic screening of human disease). They represent excellent experimental models for addressing studies on development, morphology, physiology and behavior function in other related species, as well as informative analysis of conservation and diversity. Although less complex, fish share many anatomical and physiological features with mammals, including humans, which make them an important complement to research in mammalian models. In this review we describe and compare the most relevant anatomical features of the most used teleostean species in research, to be taken into consideration when selecting an animal model: zebrafish (Danio rerio), medaka (Oryzias latypes), the turquoise killifish (Nothobranchius furzeri), and goldfish (Carassius auratus). Zebrafish and medaka are the mainstream models for genetic manipulability and studies on developmental biology; the turquoise killifish is an excellent model for aging research; goldfish has been largely employed for neuroendocrine studies.

Morphological analysis of the early development of telencephalic and diencephalic gonadotropin-releasing hormone neuronal systems in enhanced green fluorescent protein-expressing transgenic medaka lines.

Teleosts possess two or three paralogs of gonadotropin-releasing hormone (GnRH) genes: gnrh1, gnrh2, and gnrh3. Some species have lost the gnrh1 and/or gnrh3 genes, whereas gnrh2 has been completely conserved in the teleost species analyzed to date. In most teleosts that possess gnrh1, GnRH1 peptide is the authentic GnRH that stimulates gonadotropin release, whereas GnRH2 and GnRH3, if present, are neuromodulatory. Progenitors of GnRH1 and GnRH3 neurons originate from olfactory placodes and migrate to their destination during early development. However, because of the relatively low affinity/specificity of generally available antibodies that recognize GnRH1 or GnRH3, labeling of these neurons has only been possible using genetic manipulation. We used a model teleost, medaka, which possesses all three paralogous gnrh genes, to analyze development of forebrain GnRH neurons composed of GnRH1 and GnRH3 neurons. Here, we newly generated transgenic medaka lines that express enhanced green fluorescent protein under the control of promoters for gnrh1 or gnrh3, to detect GnRH neurons and facilitate immunohistochemical analysis of the neuronal morphology. We used a combination of immunohistochemistry and three-dimensional confocal microscopy image reconstructions to improve identification of neurites from GnRH1 or GnRH3 neuronal populations with greater precision. This led us to clearly identify the hypophysiotropic innervation of GnRH1 neurons residing in the ventral preoptic area (vPOA) from as early as 10 days post hatching. Furthermore, these analyses also revealed retinopetal projections of nonhypophysiotropic GnRH1 neurons in vPOA, prominent during early developmental stages, and multiple populations of GnRH3 neurons with different origins and migratory pathways.

The Parapineal Is Incorporated into the Habenula during Ontogenesis in the Medaka Fish.

The parapineal is present in many teleost families, while it is absent in several others. To find out why the parapineal is absent at adult stages in the latter families, the development of the epithalamus was examined in the medaka fish (Oryzias latipes). For this purpose, a green fluorescent protein-transgenic medaka line, in which the pineal complex (pineal and parapineal) is visible fluorescently, was used. We found that a distinct parapineal was present in the roof plate at early developmental stages. Subsequently, however, the parapineal and the associated roof plate began to be incorporated into the habenula between embryonic stages 28 and 29. Between embryonic stages 29 and 30, the entire parapineal was incorporated into the habenula. That is, the parapineal became a small caudomedial region (termed the 'parapineal domain') within the left habenula in the majority of embryos, resulting in the left-sided asymmetry of the epithalamus. Thereby the left habenula became larger and more complex than its right counterpart. In the minority of embryos, the parapineal was incorporated into the right habenula or into the habenulae on both sides. In the majority of embryos, the parapineal domain projected a fiber bundle to a subnucleus (termed the 'rostromedial subnucleus') in the left habenula. The rostromedial subnucleus sent axons, through the left fasciculus retroflexus, to the rostral region of the left half of the interpeduncular nucleus. We further found that the ratio of the left-sided phenotype was temperature dependent and decreased in embryos raised at a high temperature. The present study is the first demonstration that the supposed lack of a distinct parapineal in adult teleost fishes is due to ontogenetic incorporation into the habenula.

Crumbling under pressure.

In order for an organism to maintain its form, it must be able to withstand physical perturbation, including the pull of gravity. A recent study in Nature from Porazinski and colleagues (2015) suggests that mechanisms promoting tissue tension are critical to resist the Earth's downward pull.