Temporal dynamics of courtship and spawning in medaka under laboratory conditions revealed by 24-hour video monitoring: comparisons with natural environments

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However, the ecological validity of these findings is often uncertain when natural behaviors remain understudied. Medaka ( Oryzias latipes ) is a widely used model in reproductive and behavioral research, but the timing of its spawning in natural settings has rarely been directly observed. Recent fieldwork suggested that medaka spawn several hours before sunrise, contrasting with the common laboratory-based assumption that spawning occurs within an hour before or after light exposure. In this study, we conducted continuous 24-h video recordings of medaka pairs under controlled laboratory conditions (14L:10D photoperiod) to quantify diel variations in courtship and spawning behavior. Spawning occurred mostly between 08:00 and 11:00, peaking just after lights-on (08:00). Courtship behavior began during the dark period, increased before lights-on, and peaked between 07:00 and 09:00. These patterns mirrored field observations but showed a consistent temporal lag under laboratory conditions. This shift likely reflects differences in photoperiod timing, lack of gradual light transitions, and stable water temperatures. Our findings underscore the importance of designing experimental protocols informed by ecological dynamics, ensuring more accurate behavioral inferences in medaka and other model organisms. Earth and environmental sciences/Ecology/Freshwater ecology Biological sciences/Zoology/Animal behaviour Biological sciences/Ecology/Behavioural ecology Spawning Courtship Model organism Medaka Oryzias latipes Video observation Figures Figure 1 Introduction Model organisms play a central role in elucidating biological mechanisms, often through studies conducted in highly controlled laboratories. However, the extent to which such findings generalize to natural environments remains uncertain, as ecological information on many model species remains limited[ 1 ]. This disconnect raises critical concerns regarding the ecological validity and scientific rigor of laboratory-based research. Environmental differences between laboratory and field conditions can induce substantial shifts in behavioral and physiological responses. The relevance of the observed patterns to natural systems becomes questionable when experimental settings fail to replicate key ecological variables[ 2 ]. In response, there is a growing recognition of the need for comparative studies in which behavioral and physiological traits are evaluated in the context of laboratory and field conditions[ 3 – 5 ]. When transferring insights from model organism research to applied fields, such as medicine and conservation biology, understanding the differences between laboratory and field environments ensures the robustness of scientific evidence. For example, in zebrafish, morphological and behavioral characteristics differ significantly between laboratory and natural environments, which could have serious implications for the generalization and application of research findings[ 5 – 8 ]. Additionally, owing to domestication and laboratory breeding, other organisms have shown behaviors that differ from those shown in natural environments[ 9 – 11 ]. Despite its long history as a model organism, the ecological biology of medaka ( Oryzias latipes ), particularly its reproductive ecology, remains poorly understood in natural settings[ 4 ]. Medaka are widely used in physiology, genetics, developmental biology, behavioral science, and biomedical research because of their small body size, ease of rearing, pronounced sexual dimorphism, short generation time, transparent eggs, and a compact genome[ 12 , 13 ]. However, these features are the products of natural and sexual selection, and their functional significance cannot be fully understood without observations under ecologically relevant conditions. In the wild, medaka occupy still or slow-flowing freshwater habitats, such as rice paddies, ponds, and irrigation canals across Japan. They typically live for approximately one year and reproduce from early summer to autumn[ 14 – 17 ]. In laboratory settings, spawning occurs in isolated male–female pairs, with eggs externally fertilized and temporarily retained on the female abdomen before being deposited onto substrates such as aquatic plants. Males engage in multiple daily spawning events, whereas females typically spawn once daily[ 18 , 19 ]. Owing to the ease of observation, numerous studies on mate choice[ 20 – 22 ], mate guarding behavior[ 23 – 25 ], and alternative reproductive tactics[ 26 – 28 ] have been conducted in laboratory settings. The results of conventional laboratory studies have suggested that medaka typically initiate mating within one hour before or after the onset of light[ 29 – 32 ]. However, these conclusions were drawn largely from indirect methods, such as developmental staging of fertilized eggs, or visual observations restricted to daylight hours, leaving the exact timing of spawning initiation unresolved. Although laboratory studies have shown that ovulation in females is completed at night[ 32 ], recent work has demonstrated that general activity levels begin to rise several hours before the onset of light[ 33 ]. The results of our recent video-based observations conducted in the field and semi-natural settings have challenged this conventional understanding. In the field, post-spawning females were recorded as early as midnight, well before sunrise at approximately 05:00 h[ 34 ]. Similarly, under semi-outdoor conditions, peak spawning occurred between 02:00 and 04:00, prior to sunrise at approximately 04:45[ 35 ]. These findings strongly suggest that spawning in natural environments begins several hours before sunrise, in contrast to laboratory-based assumptions. These findings prompt a critical reassessment of the timing and context of reproductive behavior in medaka and underscore the importance of integrating laboratory and field perspectives. Quantifying behavioral differences between these settings is essential for optimizing experimental protocols that better reflect natural conditions and for designing studies that elicit ecologically meaningful behaviors. To date, there are no published reports of the precise timing of spawning initiation in medaka in laboratory aquariums, and systematic comparisons with field data are limited. Bridging this gap is particularly crucial in model organism research, in which both scientific rigor and ecological validity must be maintained to ensure meaningful inferences in both basic and applied sciences. Accordingly, we designed the present study to investigate the temporal dynamics of courtship and spawning in laboratory-reared medaka; 24-h behavioral observations were carried out to (1) identify female spawning initiation time and (2) elucidate temporal changes in the intensity of male courtship behavior. By comparing the results obtained in the present study with those of our previous field and semi-field observational studies[ 34 ], we aimed to clarify the differences in courtship and spawning behaviors between laboratory and (semi-) natural environments and examine the factors that affect these differences. Ultimately, the results of this study will contribute to a more ecologically grounded interpretation of laboratory findings and inform the development of experimental protocols that incorporate natural temporal patterns. Methods Study fish and rearing The medaka used in the experiment were purchased from the same local pet shop as in Kondo and Awata, 2025 (in press)[ 35 ]. These individuals were maintained under the following conditions for one month before being used in experiments: Eight breeding tanks (90.5 × 60.5 × 21.0 cm, length × width × height) were set up in the laboratory, with approximately 100 individuals housed in each tank. Water temperature was maintained at 26 ± 1°C, and a 14L:10D photoperiod was used (lights on at 08:00 and off at 22:00). Fish were fed Tetramin (Tetra, Melle, Germany) three times per day. Spawning activity was monitored daily during the acclimation period. Experimental settings and video recording Experiments were conducted from February 5 to 15, 2025, under the same temperature and photoperiod conditions as described above, following the protocol described by Kondo and Awata (in press)[ 35 ]. Experimental tanks (22.5 × 15.8 × 5.5 cm; L × W × H) were filled with 950 mL of water, resulting in a depth of approximately 2.7 cm. Each night, between 20:00 and 21:00, one male and one female from each breeding tank were chosen and introduced into the experimental tank at 21:00. A total of 35 male–female pairs (2–4 pairs per day) were tested (Supplementary Table S1 ). Continuous 24-h video recording was carried out using an AURORA PRO C011300 camera (SiOnyx, Beverly, MA, USA) equipped with a 512 GB SD card (SanDisk, Milpitas, CA, USA). Nighttime recordings (22:00–08:00) were conducted in infrared mode using 940 nm illumination (EnergyPower, Hong Kong, China), which is outside the visual sensitivity range of medaka and does not affect behavior[ 36 – 39 ]. Daytime recordings (08:00–22:00 h) were performed in standard mode. No food was provided during the experiments. At the end of the 24-h period (21:00 the following day), the fish were anesthetized by immersion in a solution of FA100 (DS Pharma Animal Health, Osaka, Japan) diluted 1:2,000 (0.25 mL per 500 mL of water), and body mass was measured with an electronic balance (HT-120, A&D, Tokyo). After measurements, the individuals were returned to their original breeding tanks. Each individual was examined only once. Behavioral analysis The video data were analysed using ELAN version 6.8 annotation software. To assess diel behavioral variation, 10-min video clips were extracted from the 20–30-min segment of each hour, yielding 24 clips per pair of medaka. Previous studies have shown that spawning behavior in medaka typically follows a stereotypical sequence[ 40 , 41 ]: (1) following, in which the male follows the female; (2) quick circle, in which the male swims rapidly around the female; (3) wrapping, in which the male encircles the female with his dorsal and anal fins; (4) egg and sperm release; and (5) leaving. After spawning, females carry eggs attached to their abdomen and deposit them on aquatic vegetation. Behavioral sequences during spawning, including egg and sperm release, were classified according to established criteria, and spawning time was defined as the moment of initiation of the spawning act, which was identified visually from the recordings[ 35 , 42 ]. Furthermore, based on previous work[ 34 , 35 ], we quantified two courtship behaviors from each 10-minute clip: (1) the total duration of the following and (2) the frequency of quick circles. Statistical analysis The mean body mass of males was 0.29 g (SD = 0.01; range: 0.21–0.39; n = 35), and that of females was 0.31 g (SD = 0.01; range: 0.23–0.50; n = 35; see Supplementary Table S1 ). All statistical analyses were conducted using R version 4.4.1 (R Core Team 2024)[ 43 ]. To estimate the peak spawning timing, we fitted a gamma distribution to the observed spawning events. Temporal changes in courtship behavior (following duration and quick-circle frequency) were analysed using generalized additive mixed models (GAMMs). Model significance was assessed using likelihood ratio tests, with statistical significance defined as p < 0.05. The response variable was the total duration of the following observed in each 10-min video segment: Time of day (hours) was included as a fixed effect, and male ID was specified as a random effect. The model was fitted to a gaussian distribution. With respect to quick-circle frequency, the response variable was the number of quick-circle displays per 10-min segment. Time of day was again included as a fixed effect, and male ID was included as a random effect. The model was fitted using a negative binomial distribution to account for overdispersion. Results Spawning behavior A total of 35 spawning events were recorded between 07:23 and 13:47 (Supplementary Movie S1). The majority (25 of 35 events, 71%) occurred between 08:00 and 11:00, with a distinct peak shortly after lights were turned on at 08:00 (Fig. 1 a; Supplementary Table S1 ). Twelve events (34%) occurred within one hour of or after lights-on. In total, 31 spawning events (89%) occurred after light onset, whereas only four events (11%) occurred beforehand. Courtship behavior To examine diel variations in courtship behavior, we analysed 24-h recordings from all 35 spawning pairs. “Following” was observed consistently throughout the day, with an average duration of 181.8 ± 6.0 s per 10-min video segment (n = 840 observations from 35 males; mean ± SE; Supplementary Movie S2). The following duration increased during the dark period, peaked between 07:00 and 09:00, and declined thereafter (GAMM: deviance = 4075648; df = 8.75; p < 0.0001; Fig. 1 b). “Quick circle” occurred 476 times in total, averaging 0.57 ± 0.07 occurrences per video segment (n = 840; Supplementary Movie S3). The temporal pattern of quick circle mirrored that of following; it increased prior to light onset, peaked between 07:00 and 09:00, and declined substantially after 12:00 (GAMM: deviance = 102.56; df = 7.71; p < 0.0001; Fig. 1 c). Notably, most post-mating males did not perform quick circle after 12:00. Discussion Reproductive timing is a key ecological trait that significantly influences fitness. In medaka, spawning has been previously reported to occur within one hour before or after lights on[ 29 – 32 ], although these conclusions are based on indirect observations rather than direct monitoring under dark conditions. In the present study, we provide the first direct evidence of spawning initiation and courtship rhythms under controlled laboratory conditions using continuous video surveillance. Our findings generally align with previous reports that spawning primarily occurs shortly after lights-on; however, we also discovered that courtship behavior begins during the dark period. This previously undocumented nocturnal initiation of courtship behavior represents a significant revision of the conventional understanding of medaka reproductive behaviors in laboratory settings. Notably, our 24-h continuous video monitoring methodology revealed that medaka initiate courtship activities during the dark period, a phenomenon not previously reported in laboratory settings. Whereas it had been reported previously that the general activity level of the medaka increased several hours before lights-on[ 33 ], our detailed behavioral analysis has, for the first time, identified specific reproductive behaviors driving the pre-dawn period. This methodological advancement allowed us to document the complete chronological sequence of reproductive behaviors, providing a behavioral foundation for previously unexplained activity patterns. Moreover, prior research has shown that medaka can spawn in the dark[ 44 ] and that olfactory cues are integral to the reproductive process[ 41 ]. The occurrence of nocturnal courtship suggests that medaka can detect and respond to reproductive cues via non-visual modalities, likely by relying on olfaction. A key finding of this study is the consistent 3- to 4-h delay in the timing of courtship and spawning behaviors in laboratory settings compared to field and semi-natural environments, despite similarities in overall behavioral patterns. Several factors may underlie these observed temporal shifts. The most immediate explanation involves the discrepancy between the artificial light cycle in the laboratory (lights on at 08:00 and lights off at 22:00) and the natural timing of sunrise and sunset. As medaka behavioral rhythms are highly sensitive to photoperiod, entrainment into this artificial schedule likely influences the timing of reproductive behaviors[ 16 , 44 , 45 ]. Given the known role of thyroid-stimulating hormones in the photoperiodic regulation of seasonal reproduction in birds, mammals[ 46 – 48 ], and fish[ 49 ], such shifts in photoperiodic signaling could modulate reproductive timing in medaka as well. However, photoperiod differences alone may not fully account for temporal shifts. In natural environments, ambient light intensity gradually increases during the pre-dawn period, whereas laboratory lighting typically operates on an abrupt on/off cycle. This qualitative difference may have affected the onset of behavioral expression. Diurnal temperature fluctuations may also have contributed to this phenomenon. While natural habitats exhibit temperature minima during the night and early morning, our laboratory setup maintained water temperature at a constant 26 ± 1°C. As temperature influences both physiological processes and behavioral rhythms across taxa, it is plausible that thermal stability affects spawning timing. Given that temperature has also been identified as a critical cue in the seasonal reproduction of bony fishes[ 50 , 51 ], even in endothermic species[ 52 – 54 ], the absence of naturalistic thermal variability in laboratory settings may further distort reproductive timing. This study had several limitations. First, our findings were based solely on a single laboratory strain (Himedaka), and caution should be exercised when extrapolating to wild populations. Secondly, although we identified the timing of spawning, the exact timing of ovulation was not directly measured. Although earlier work suggests that ovulation is completed at night[ 32 ], the precise timing under laboratory conditions remains unknown. Future studies employing fine-scale hormonal or physiological monitoring are required to clarify the relationship between ovulation and courtship. The adaptive value of nocturnal reproduction, such as avoidance of visual predators, warrants further investigation. Assessing the predation risk on medaka and their eggs across diel cycles in natural environments would provide insights into the potential selective pressures shaping reproductive timing. Most research on model organisms has been conducted in laboratory settings, enabling the precise control of environmental variables and facilitating fundamental discoveries in biology[ 1 ]. However, these controlled conditions often omit key ecological factors, leading to gaps in our understanding of naturalistic behavior. A number of organisms have shown behaviors differing from those in natural environments, due to domestication and laboratory breeding[ 9 – 11 ]. The 3- to 4-h delay in reproductive timing observed in this study exemplifies such a gap between laboratory and field conditions. These temporal mismatches may have far-reaching implications for experimental design, particularly the investigation of diel variations in hormone secretion, gene expression, and reproductive behavior in medaka. Our findings suggest that sampling schedules and analytical frameworks should account for field-based temporal dynamics to avoid systematic bias. By showing that courtship initiation during the dark period occurs in both laboratory and field environments, albeit with a consistent time lag, this study highlights the need to bridge experimental and ecological contexts. Developing experimental protocols that incorporate more naturalistic environmental features, such as gradual light transitions (e.g., pre-dawn and dawn), temperature fluctuations, and field-aligned photoperiods, will allow researchers to elicit more ecologically valid behaviors. Such integrative approaches offer a path toward unifying the mechanistic precision of laboratory science with the ecological realism of field biology, thereby improving the robustness and interpretability of model organism research. Declarations Acknowledgements We extend our gratitude to the members of the Laboratory of Animal Sociology at the Osaka Metropolitan University for their valuable support throughout this study. We also acknowledge Editage (www.editage.jp) for providing English language editing services. Author Contributions Y. Kondo, R. K., Y. Kobayashi and S. A. designed the study. Y. Kondo conducted the aquarium experiments and collected the data. Y. Kondo and S. A. performed the analyses. Y. Kondo, R. K., Y. Kobayashi and S. A. wrote and approved the final manuscript for publication. Data Availability Data are included as Supplementary Information. Ethical Note All experimental procedures were conducted in accordance with the ARRIVE guidelines[55] and with the approval of Osaka Metropolitan University (Approval Number: S0092). 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Supplementary Files SupplementaryMaterials.docx MovieS1.mp4 MovieS2.mp4 MovieS3.mp4 TableS2.csv Cite Share Download PDF Status: Published Journal Publication published 07 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Jun, 2025 Reviews received at journal 10 Jun, 2025 Reviewers agreed at journal 08 Jun, 2025 Reviews received at journal 03 Jun, 2025 Reviewers agreed at journal 27 May, 2025 Reviewers agreed at journal 27 May, 2025 Reviewers agreed at journal 26 May, 2025 Reviewers invited by journal 26 May, 2025 Editor assigned by journal 26 May, 2025 Editor invited by journal 21 May, 2025 Submission checks completed at journal 09 May, 2025 First submitted to journal 09 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6610344","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":454752706,"identity":"d14d71b8-4588-4de8-ba77-1af5e412f367","order_by":0,"name":"Yuki Kondo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYHACZoaEA0DqAAPjAyDFw0eKFmYDkBY2orQwQLSwSYC4BLUYHO8xNnhwxiaP73iPWeXXHDsZNgbmh49u4NNy5oxxQsKNtGLJM2fMbstuSwY6jM3YOAeflhs5xgcSPhxO3HAjx+y25DZmoBYeNmmitRRLbqsnTgvQYRAtjB+3HSasRfLMsWKDhDNpiTOBDGnGbcd52JgJ+IXvePNmyR/HbBL7jjdv/PhzW7U9P3vzw8f4tCgcQOIw84BJPMpBQL4BicP4g4DqUTAKRsEoGJkAAKy2UDKeh5LoAAAAAElFTkSuQmCC","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":true,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Kondo","suffix":""},{"id":454752707,"identity":"168dec29-e993-445b-ae55-4c0ca738ca55","order_by":1,"name":"Ryotaro Kobayashi","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Ryotaro","middleName":"","lastName":"Kobayashi","suffix":""},{"id":454752708,"identity":"c9a38c27-e9f8-4778-8614-3b9692ca43ff","order_by":2,"name":"Yuya Kobayashi","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Yuya","middleName":"","lastName":"Kobayashi","suffix":""},{"id":454752709,"identity":"76f2ecb8-f2c0-4b5c-bb60-a40bd79026df","order_by":3,"name":"Satoshi Awata","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Awata","suffix":""}],"badges":[],"createdAt":"2025-05-07 09:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6610344/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6610344/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-11082-y","type":"published","date":"2025-08-07T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82484889,"identity":"6401a473-321f-47c8-9db8-1b70f1ebd012","added_by":"auto","created_at":"2025-05-12 04:59:21","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471522,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Frequency distribution of spawning events observed in each hour over 24 h in medaka, \u003cem\u003eOryzias latipes\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e = 35). The peak of spawning events was determined using gamma distribution fitting. (b) and (c) Changes in courtship behaviors from 21:00 to 20:00 over 24 h. (b) The total following duration (sec/10 min) and (c) the number of quick-circle behaviors (number / 10 min). Each plot (green circle: pre-mating male; red triangle: post-mating male) signifies the observed values from the analysed videos. The regression curves were based on the generalized additive mixed models (GAMMs) using all data (\u003cem\u003en\u003c/em\u003e = 840 observations from 35 males), and the shading indicates the 95% confidence intervals.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/3422dbd83326116f922d317c.jpeg"},{"id":88814146,"identity":"3f354010-5b41-4729-a1a5-bd77474fdcc9","added_by":"auto","created_at":"2025-08-11 16:07:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1051945,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/0bb8ba7c-a508-4ef3-8fc0-677487c26ecc.pdf"},{"id":82484529,"identity":"f5c92c15-0c88-486a-a1f6-8de5e49076ff","added_by":"auto","created_at":"2025-05-12 04:51:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23717,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/614059532be976cfb68bb040.docx"},{"id":82484532,"identity":"446b2d75-da93-4c34-ac59-f591f598c764","added_by":"auto","created_at":"2025-05-12 04:51:21","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1825979,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/541cecf8b827b92768950a5c.mp4"},{"id":82484890,"identity":"eca4468e-d81f-4693-9867-908e069096c9","added_by":"auto","created_at":"2025-05-12 04:59:21","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":455028,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/bbc3bd372e1a7db407794b2e.mp4"},{"id":82484893,"identity":"b11e5f93-2ea8-4524-92b1-dd1a94e5e041","added_by":"auto","created_at":"2025-05-12 04:59:21","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":539068,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/dbd6ea16c9d59464ebf5ecfd.mp4"},{"id":82484539,"identity":"0161d73a-b7ac-44fc-8478-25800db71618","added_by":"auto","created_at":"2025-05-12 04:51:21","extension":"csv","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":22682,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.csv","url":"https://assets-eu.researchsquare.com/files/rs-6610344/v1/614f1bceb559fd59e859881f.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Temporal dynamics of courtship and spawning in medaka under laboratory conditions revealed by 24-hour video monitoring: comparisons with natural environments","fulltext":[{"header":"Introduction","content":"\u003cp\u003eModel organisms play a central role in elucidating biological mechanisms, often through studies conducted in highly controlled laboratories. However, the extent to which such findings generalize to natural environments remains uncertain, as ecological information on many model species remains limited[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This disconnect raises critical concerns regarding the ecological validity and scientific rigor of laboratory-based research. Environmental differences between laboratory and field conditions can induce substantial shifts in behavioral and physiological responses. The relevance of the observed patterns to natural systems becomes questionable when experimental settings fail to replicate key ecological variables[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In response, there is a growing recognition of the need for comparative studies in which behavioral and physiological traits are evaluated in the context of laboratory and field conditions[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen transferring insights from model organism research to applied fields, such as medicine and conservation biology, understanding the differences between laboratory and field environments ensures the robustness of scientific evidence. For example, in zebrafish, morphological and behavioral characteristics differ significantly between laboratory and natural environments, which could have serious implications for the generalization and application of research findings[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, owing to domestication and laboratory breeding, other organisms have shown behaviors that differ from those shown in natural environments[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite its long history as a model organism, the ecological biology of medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e), particularly its reproductive ecology, remains poorly understood in natural settings[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Medaka are widely used in physiology, genetics, developmental biology, behavioral science, and biomedical research because of their small body size, ease of rearing, pronounced sexual dimorphism, short generation time, transparent eggs, and a compact genome[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, these features are the products of natural and sexual selection, and their functional significance cannot be fully understood without observations under ecologically relevant conditions.\u003c/p\u003e \u003cp\u003eIn the wild, medaka occupy still or slow-flowing freshwater habitats, such as rice paddies, ponds, and irrigation canals across Japan. They typically live for approximately one year and reproduce from early summer to autumn[\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In laboratory settings, spawning occurs in isolated male\u0026ndash;female pairs, with eggs externally fertilized and temporarily retained on the female abdomen before being deposited onto substrates such as aquatic plants. Males engage in multiple daily spawning events, whereas females typically spawn once daily[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Owing to the ease of observation, numerous studies on mate choice[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], mate guarding behavior[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and alternative reproductive tactics[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] have been conducted in laboratory settings.\u003c/p\u003e \u003cp\u003eThe results of conventional laboratory studies have suggested that medaka typically initiate mating within one hour before or after the onset of light[\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, these conclusions were drawn largely from indirect methods, such as developmental staging of fertilized eggs, or visual observations restricted to daylight hours, leaving the exact timing of spawning initiation unresolved. Although laboratory studies have shown that ovulation in females is completed at night[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], recent work has demonstrated that general activity levels begin to rise several hours before the onset of light[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The results of our recent video-based observations conducted in the field and semi-natural settings have challenged this conventional understanding. In the field, post-spawning females were recorded as early as midnight, well before sunrise at approximately 05:00 h[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Similarly, under semi-outdoor conditions, peak spawning occurred between 02:00 and 04:00, prior to sunrise at approximately 04:45[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These findings strongly suggest that spawning in natural environments begins several hours before sunrise, in contrast to laboratory-based assumptions.\u003c/p\u003e \u003cp\u003eThese findings prompt a critical reassessment of the timing and context of reproductive behavior in medaka and underscore the importance of integrating laboratory and field perspectives. Quantifying behavioral differences between these settings is essential for optimizing experimental protocols that better reflect natural conditions and for designing studies that elicit ecologically meaningful behaviors. To date, there are no published reports of the precise timing of spawning initiation in medaka in laboratory aquariums, and systematic comparisons with field data are limited. Bridging this gap is particularly crucial in model organism research, in which both scientific rigor and ecological validity must be maintained to ensure meaningful inferences in both basic and applied sciences.\u003c/p\u003e \u003cp\u003eAccordingly, we designed the present study to investigate the temporal dynamics of courtship and spawning in laboratory-reared medaka; 24-h behavioral observations were carried out to (1) identify female spawning initiation time and (2) elucidate temporal changes in the intensity of male courtship behavior. By comparing the results obtained in the present study with those of our previous field and semi-field observational studies[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we aimed to clarify the differences in courtship and spawning behaviors between laboratory and (semi-) natural environments and examine the factors that affect these differences. Ultimately, the results of this study will contribute to a more ecologically grounded interpretation of laboratory findings and inform the development of experimental protocols that incorporate natural temporal patterns.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy fish and rearing\u003c/h2\u003e \u003cp\u003eThe medaka used in the experiment were purchased from the same local pet shop as in Kondo and Awata, 2025 (in press)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These individuals were maintained under the following conditions for one month before being used in experiments: Eight breeding tanks (90.5 \u0026times; 60.5 \u0026times; 21.0 cm, length \u0026times; width \u0026times; height) were set up in the laboratory, with approximately 100 individuals housed in each tank. Water temperature was maintained at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and a 14L:10D photoperiod was used (lights on at 08:00 and off at 22:00). Fish were fed Tetramin (Tetra, Melle, Germany) three times per day. Spawning activity was monitored daily during the acclimation period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental settings and video recording\u003c/h3\u003e\n\u003cp\u003eExperiments were conducted from February 5 to 15, 2025, under the same temperature and photoperiod conditions as described above, following the protocol described by Kondo and Awata (in press)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Experimental tanks (22.5 \u0026times; 15.8 \u0026times; 5.5 cm; L \u0026times; W \u0026times; H) were filled with 950 mL of water, resulting in a depth of approximately 2.7 cm.\u003c/p\u003e \u003cp\u003eEach night, between 20:00 and 21:00, one male and one female from each breeding tank were chosen and introduced into the experimental tank at 21:00. A total of 35 male\u0026ndash;female pairs (2\u0026ndash;4 pairs per day) were tested (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eContinuous 24-h video recording was carried out using an AURORA PRO C011300 camera (SiOnyx, Beverly, MA, USA) equipped with a 512 GB SD card (SanDisk, Milpitas, CA, USA). Nighttime recordings (22:00\u0026ndash;08:00) were conducted in infrared mode using 940 nm illumination (EnergyPower, Hong Kong, China), which is outside the visual sensitivity range of medaka and does not affect behavior[\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Daytime recordings (08:00\u0026ndash;22:00 h) were performed in standard mode. No food was provided during the experiments.\u003c/p\u003e \u003cp\u003eAt the end of the 24-h period (21:00 the following day), the fish were anesthetized by immersion in a solution of FA100 (DS Pharma Animal Health, Osaka, Japan) diluted 1:2,000 (0.25 mL per 500 mL of water), and body mass was measured with an electronic balance (HT-120, A\u0026amp;D, Tokyo). After measurements, the individuals were returned to their original breeding tanks. Each individual was examined only once.\u003c/p\u003e\n\u003ch3\u003eBehavioral analysis\u003c/h3\u003e\n\u003cp\u003eThe video data were analysed using ELAN version 6.8 annotation software. To assess diel behavioral variation, 10-min video clips were extracted from the 20\u0026ndash;30-min segment of each hour, yielding 24 clips per pair of medaka. Previous studies have shown that spawning behavior in medaka typically follows a stereotypical sequence[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]: (1) following, in which the male follows the female; (2) quick circle, in which the male swims rapidly around the female; (3) wrapping, in which the male encircles the female with his dorsal and anal fins; (4) egg and sperm release; and (5) leaving. After spawning, females carry eggs attached to their abdomen and deposit them on aquatic vegetation. Behavioral sequences during spawning, including egg and sperm release, were classified according to established criteria, and spawning time was defined as the moment of initiation of the spawning act, which was identified visually from the recordings[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Furthermore, based on previous work[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], we quantified two courtship behaviors from each 10-minute clip: (1) the total duration of the following and (2) the frequency of quick circles.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe mean body mass of males was 0.29 g (SD\u0026thinsp;=\u0026thinsp;0.01; range: 0.21\u0026ndash;0.39; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;35), and that of females was 0.31 g (SD\u0026thinsp;=\u0026thinsp;0.01; range: 0.23\u0026ndash;0.50; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;35; see Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All statistical analyses were conducted using R version 4.4.1 (R Core Team 2024)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo estimate the peak spawning timing, we fitted a gamma distribution to the observed spawning events. Temporal changes in courtship behavior (following duration and quick-circle frequency) were analysed using generalized additive mixed models (GAMMs). Model significance was assessed using likelihood ratio tests, with statistical significance defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The response variable was the total duration of the following observed in each 10-min video segment: Time of day (hours) was included as a fixed effect, and male ID was specified as a random effect. The model was fitted to a gaussian distribution. With respect to quick-circle frequency, the response variable was the number of quick-circle displays per 10-min segment. Time of day was again included as a fixed effect, and male ID was included as a random effect. The model was fitted using a negative binomial distribution to account for overdispersion.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpawning behavior\u003c/h2\u003e \u003cp\u003eA total of 35 spawning events were recorded between 07:23 and 13:47 (Supplementary Movie S1). The majority (25 of 35 events, 71%) occurred between 08:00 and 11:00, with a distinct peak shortly after lights were turned on at 08:00 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Twelve events (34%) occurred within one hour of or after lights-on. In total, 31 spawning events (89%) occurred after light onset, whereas only four events (11%) occurred beforehand.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCourtship behavior\u003c/h3\u003e\n\u003cp\u003eTo examine diel variations in courtship behavior, we analysed 24-h recordings from all 35 spawning pairs. \u0026ldquo;Following\u0026rdquo; was observed consistently throughout the day, with an average duration of 181.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 s per 10-min video segment (n\u0026thinsp;=\u0026thinsp;840 observations from 35 males; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE; Supplementary Movie S2). The following duration increased during the dark period, peaked between 07:00 and 09:00, and declined thereafter (GAMM: deviance\u0026thinsp;=\u0026thinsp;4075648; df\u0026thinsp;=\u0026thinsp;8.75; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e\u0026ldquo;Quick circle\u0026rdquo; occurred 476 times in total, averaging 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 occurrences per video segment (n\u0026thinsp;=\u0026thinsp;840; Supplementary Movie S3). The temporal pattern of quick circle mirrored that of following; it increased prior to light onset, peaked between 07:00 and 09:00, and declined substantially after 12:00 (GAMM: deviance\u0026thinsp;=\u0026thinsp;102.56; df\u0026thinsp;=\u0026thinsp;7.71; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Notably, most post-mating males did not perform quick circle after 12:00.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eReproductive timing is a key ecological trait that significantly influences fitness. In medaka, spawning has been previously reported to occur within one hour before or after lights on[\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], although these conclusions are based on indirect observations rather than direct monitoring under dark conditions. In the present study, we provide the first direct evidence of spawning initiation and courtship rhythms under controlled laboratory conditions using continuous video surveillance. Our findings generally align with previous reports that spawning primarily occurs shortly after lights-on; however, we also discovered that courtship behavior begins during the dark period. This previously undocumented nocturnal initiation of courtship behavior represents a significant revision of the conventional understanding of medaka reproductive behaviors in laboratory settings.\u003c/p\u003e \u003cp\u003eNotably, our 24-h continuous video monitoring methodology revealed that medaka initiate courtship activities during the dark period, a phenomenon not previously reported in laboratory settings. Whereas it had been reported previously that the general activity level of the medaka increased several hours before lights-on[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], our detailed behavioral analysis has, for the first time, identified specific reproductive behaviors driving the pre-dawn period. This methodological advancement allowed us to document the complete chronological sequence of reproductive behaviors, providing a behavioral foundation for previously unexplained activity patterns. Moreover, prior research has shown that medaka can spawn in the dark[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and that olfactory cues are integral to the reproductive process[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The occurrence of nocturnal courtship suggests that medaka can detect and respond to reproductive cues via non-visual modalities, likely by relying on olfaction.\u003c/p\u003e \u003cp\u003eA key finding of this study is the consistent 3- to 4-h delay in the timing of courtship and spawning behaviors in laboratory settings compared to field and semi-natural environments, despite similarities in overall behavioral patterns. Several factors may underlie these observed temporal shifts. The most immediate explanation involves the discrepancy between the artificial light cycle in the laboratory (lights on at 08:00 and lights off at 22:00) and the natural timing of sunrise and sunset. As medaka behavioral rhythms are highly sensitive to photoperiod, entrainment into this artificial schedule likely influences the timing of reproductive behaviors[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Given the known role of thyroid-stimulating hormones in the photoperiodic regulation of seasonal reproduction in birds, mammals[\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and fish[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], such shifts in photoperiodic signaling could modulate reproductive timing in medaka as well.\u003c/p\u003e \u003cp\u003eHowever, photoperiod differences alone may not fully account for temporal shifts. In natural environments, ambient light intensity gradually increases during the pre-dawn period, whereas laboratory lighting typically operates on an abrupt on/off cycle. This qualitative difference may have affected the onset of behavioral expression. Diurnal temperature fluctuations may also have contributed to this phenomenon. While natural habitats exhibit temperature minima during the night and early morning, our laboratory setup maintained water temperature at a constant 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. As temperature influences both physiological processes and behavioral rhythms across taxa, it is plausible that thermal stability affects spawning timing. Given that temperature has also been identified as a critical cue in the seasonal reproduction of bony fishes[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], even in endothermic species[\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], the absence of naturalistic thermal variability in laboratory settings may further distort reproductive timing.\u003c/p\u003e \u003cp\u003eThis study had several limitations. First, our findings were based solely on a single laboratory strain (Himedaka), and caution should be exercised when extrapolating to wild populations. Secondly, although we identified the timing of spawning, the exact timing of ovulation was not directly measured. Although earlier work suggests that ovulation is completed at night[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], the precise timing under laboratory conditions remains unknown. Future studies employing fine-scale hormonal or physiological monitoring are required to clarify the relationship between ovulation and courtship. The adaptive value of nocturnal reproduction, such as avoidance of visual predators, warrants further investigation. Assessing the predation risk on medaka and their eggs across diel cycles in natural environments would provide insights into the potential selective pressures shaping reproductive timing.\u003c/p\u003e \u003cp\u003eMost research on model organisms has been conducted in laboratory settings, enabling the precise control of environmental variables and facilitating fundamental discoveries in biology[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, these controlled conditions often omit key ecological factors, leading to gaps in our understanding of naturalistic behavior. A number of organisms have shown behaviors differing from those in natural environments, due to domestication and laboratory breeding[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The 3- to 4-h delay in reproductive timing observed in this study exemplifies such a gap between laboratory and field conditions. These temporal mismatches may have far-reaching implications for experimental design, particularly the investigation of diel variations in hormone secretion, gene expression, and reproductive behavior in medaka. Our findings suggest that sampling schedules and analytical frameworks should account for field-based temporal dynamics to avoid systematic bias.\u003c/p\u003e \u003cp\u003eBy showing that courtship initiation during the dark period occurs in both laboratory and field environments, albeit with a consistent time lag, this study highlights the need to bridge experimental and ecological contexts. Developing experimental protocols that incorporate more naturalistic environmental features, such as gradual light transitions (e.g., pre-dawn and dawn), temperature fluctuations, and field-aligned photoperiods, will allow researchers to elicit more ecologically valid behaviors. Such integrative approaches offer a path toward unifying the mechanistic precision of laboratory science with the ecological realism of field biology, thereby improving the robustness and interpretability of model organism research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the members of the Laboratory of Animal Sociology at the Osaka Metropolitan University for their valuable support throughout this study. We also acknowledge Editage (www.editage.jp) for providing English language editing services.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. Kondo, R. K., Y. Kobayashi and S. A. designed the study. Y. Kondo conducted the aquarium experiments and collected the data. Y. Kondo and S. A. performed the analyses. Y. Kondo, R. K., Y. Kobayashi and S. A. wrote and approved the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are included as Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were conducted in accordance with the ARRIVE guidelines[55] and with the approval of Osaka Metropolitan University (Approval Number: S0092). All procedures involving animals adhered to the most recent ASAB/ABS ethical guidelines and were approved by the Animal Care and Use Committee of Osaka Metropolitan University, Japan. Housing and experimental methodologies have been developed to minimize stress in medaka. After the experiment, the fish were returned to their original tanks. We did not observe any behaviors indicative of stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Japan Society for the Promotion of Science (JSPS) KAKENHI (22K20666 to Y. Kondo and 23H03868 to S.A.), the Sasakawa Scientific Research Grant from the Japan Science Society (2023-5013 and 2024-5010 to Y. Kondo), the Tokyo Zoological Park Society Wildlife Conservation Fund (to Y. Kondo), and the Kurita Water and Environment Foundation (24H083 to Y. Kondo).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHeard, E. 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The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. \u003cem\u003ePLOS Biol.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, e3000410 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spawning, Courtship, Model organism, Medaka, Oryzias latipes, Video observation","lastPublishedDoi":"10.21203/rs.3.rs-6610344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6610344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the biological phenomena in model organisms typically relies on laboratory studies. However, the ecological validity of these findings is often uncertain when natural behaviors remain understudied. Medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e) is a widely used model in reproductive and behavioral research, but the timing of its spawning in natural settings has rarely been directly observed. Recent fieldwork suggested that medaka spawn several hours before sunrise, contrasting with the common laboratory-based assumption that spawning occurs within an hour before or after light exposure. In this study, we conducted continuous 24-h video recordings of medaka pairs under controlled laboratory conditions (14L:10D photoperiod) to quantify diel variations in courtship and spawning behavior. Spawning occurred mostly between 08:00 and 11:00, peaking just after lights-on (08:00). Courtship behavior began during the dark period, increased before lights-on, and peaked between 07:00 and 09:00. These patterns mirrored field observations but showed a consistent temporal lag under laboratory conditions. This shift likely reflects differences in photoperiod timing, lack of gradual light transitions, and stable water temperatures. Our findings underscore the importance of designing experimental protocols informed by ecological dynamics, ensuring more accurate behavioral inferences in medaka and other model organisms.\u003c/p\u003e","manuscriptTitle":"Temporal dynamics of courtship and spawning in medaka under laboratory conditions revealed by 24-hour video monitoring: comparisons with natural environments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-12 04:51:16","doi":"10.21203/rs.3.rs-6610344/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-11T05:09:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T15:25:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263002305277244706439012555272337356549","date":"2025-06-08T23:54:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-04T02:21:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248974281003580558812435189656313635721","date":"2025-05-27T14:35:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282309914014670223715767361991789820269","date":"2025-05-27T09:36:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47217049834497417785533003558768381063","date":"2025-05-27T02:08:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-27T02:03:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-27T01:25:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-21T16:52:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-10T02:19:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-10T02:18:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32fba374-7831-48dc-9fd2-99ede2b0837c","owner":[],"postedDate":"May 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48350046,"name":"Earth and environmental sciences/Ecology/Freshwater ecology"},{"id":48350047,"name":"Biological sciences/Zoology/Animal behaviour"},{"id":48350048,"name":"Biological sciences/Ecology/Behavioural ecology"}],"tags":[],"updatedAt":"2025-08-11T16:01:14+00:00","versionOfRecord":{"articleIdentity":"rs-6610344","link":"https://doi.org/10.1038/s41598-025-11082-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-07 15:57:24","publishedOnDateReadable":"August 7th, 2025"},"versionCreatedAt":"2025-05-12 04:51:16","video":"","vorDoi":"10.1038/s41598-025-11082-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-11082-y","workflowStages":[]},"version":"v1","identity":"rs-6610344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6610344","identity":"rs-6610344","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}