Japanese Medaka Oryzias Latipes Research Articles

Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks’ Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

Neuropeptide Y receptor Y8b (npy8br) regulates feeding and digestion in Japanese medaka (Oryzias latipes) larvae: evidence from gene knockout.

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br-/- Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br-/- medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br-/- medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

Time-series variation in the locomotor behavior and vocal traits of Japanese medaka (Oryzias latipes) acutely exposed to organophosphorus pesticide chlorpyrifos

Organophosphorus pesticides (OPs), such as chlorpyrifos (CPF), are the most commonly used pesticides worldwide. Considering that OPs will eventually enter aquatic ecosystems due to runoff from agricultural lands, accidental leakage, and other unforeseen emergencies, monitoring water pollution of those substances is crucial for environmental protection and public health. In this study, Japanese medaka (Oryzias latipes) were exposed to CPF (0.03, 0.06, and 0.12 mg/L) for 6 h, and the time-series variations in their locomotor behavior and vocal traits were investigated. Compared with that measured before exposure, significantly changed locomotor behavior and vocal traits in Japanese medaka exposed to CPF could be observed at 4 h after exposure and thereafter, and the pattern of behavioral changes depends on the CPF concentrations. Exposure to CPF also changed the frequency-sound pressure level curve of Japanese medaka at 6 h after exposure, especially at 0.12 mg/L. Moreover, CPF exposure could significantly inhibit the acetylcholinesterase (AChE) activity in the brains and eyes of medaka, which exhibited significant correlations with the variation of locomotor behavioral and vocal traits. Considering that inhibiting the AChE activity is the primary mechanism underlying the neurobehavioral toxicity of all OPs, our finding suggested that simultaneously monitoring changes in the locomotor behavioral and vocal traits has a high potential to reflect the pollution of organophosphorus substances.

Role of Medaka (Oryzias latipes) Foxo3 in Resistance to Nervous Necrosis Virus Infection.

Upon encountering a virus, fish initiate an innate immune response, guided by IFNs. Foxo3 plays a part in the body's immune response; however, its specific role in the IFN-guided immune response in fish is yet to be clarified. In this study, we characterized foxo3 in Japanese medaka (Oryzias latipes) and examined its role in the IFN-dependent immune response upon infection with the RGNNV. The results show that the coding region of the medaka foxo3 gene is 2007 base pairs long, encoding 668 amino acids, and possesses a typical forkhead protein family structural domain. The product of this gene shares high homology with foxo3 in other fish species and is widely expressed, especially in the brain, eyes, testes, and heart. Upon RGNNV infection, foxo3-/- mutant larvae showed a lower mortality rate, and adults exhibited a significant reduction in virus replication. Moreover, the absence of foxo3 expression led to an increase in the expression of irf3, and a decrease in the expression of other IFN-related genes such as tbk1 and mapk9, implying that foxo3 may function as a negative regulator in the antiviral signaling pathway. These findings provide crucial insights for disease-resistant breeding in the aquaculture industry.

Behavioral profile alterations and predation susceptibility of Japanese medaka fish exposed to phenytoin, an antiepileptic drug

Antiepileptic drugs, such as phenytoin, are often leaked into aquatic systems through sewage facilities due to their low metabolic rate. Fish, such as the Japanese medaka (Oryzias latipes), demonstrate abnormal swimming behavior such as equilibrium abnormalities, rotational behavior, and vertical swimming, when exposed to phenytoin. Therefore, it is hypothesized that predator avoidance may be hindered. This study aimed to investigate the effects of phenytoin exposure-induced behavioral abnormalities in predator avoidance in Japanese medaka. The results showed that individuals with behavioral abnormalities had a reduced ability to avoid danger. Furthermore, the fish demonstrated a delayed recognition reaction to approaching predators. Additionally, predatory fish, such as silver pike characin (Ctenolucius hujeta), were more likely to prey upon abnormal individuals. In conclusion, the fish exposed to phenytoin demonstrated behavioral changes that increased its predation risk. This study is the first to determine the effects of behavioral abnormalities in Japanese medaka which was induced after phenytoin exposure on predator risk avoidance.

Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Effects of diphenhydramine exposure on reproduction of mature Japanese medaka (Oryzias latipes)

Diphenhydramine (DPH) is an antihistamine drug. It has been frequently detected in the environment, because it is not completely degraded in wastewater treatment plants. Recent studies have shown the adverse effects of DPH exposure to various aquatic organisms; however, its chronic effects on fish have been poorly elucidated. In this study, several pairs of mature Japanese medaka (Oryzias latipes) were exposed to DPH for a long period to determine the effects of DPH exposure on the subsequent generations, number of spawned and fertilized eggs, expression of sex-related genes, feeding behavior, embryo development, hatching rate, malformations among the hatched larvae, and mortality rate. The number of spawned eggs significantly decreased, when the parent fish were continuously exposed to 31.6 μg/L DPH for over 46 days. DPH exposure also altered the feeding behavior of medaka individuals, and increased the larval mortality rate. The effects of DPH exposure to fish may occur to some extent in the actual aquatic environment, although the risk evaluations in the field are limited.

Reproductive toxicity of DDT in the Japanese medaka fish model: Revisiting the impacts of DDT+ on female reproductive health

The organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) is an endocrine-disrupting compound (EDC) that has been banned by most countries for decades. However, it continues to be detected in nearly all humans and wildlife due to its biological and environmental persistence. The ovarian dysgenesis syndrome hypothesis speculates that exposure to EDCs during sensitive developmental windows such as early gonadal differentiation lead to reproductive disorders later in life. Yet, mechanisms by which DDT affects developing gonads remain unclear due to the inherent challenge of getting developmental exposure data from adults presenting with reproductive disease. The Japanese medaka (Oryzias latipes) is a valuable fish model for sex-specific toxicological studies due to its chromosomal sex determination, external embryonic development, short generation time, and extensively mapped genome. It is well documented that medaka exposed to DDT and its metabolites and byproducts (herein referred to as DDT+) at different developmental time points experience permanent alterations in gonadal morphology, reproductive success, and molecular and hormonal signaling. However, the overwhelming majority of studies focus primarily on functional and morphological outcomes in males and females and have rarely investigated long-term transcriptional or molecular effects. This review summarizes previous experimental findings and the state of our knowledge concerning toxic effects DDT + on reproductive development, fertility, and health in the valuable medaka model. It also identifies gaps in knowledge, emphasizing a need for more focus on molecular mechanisms of ovarian endocrine disruption using enhanced molecular tools that have become increasingly available over the past few decades. Furthermore, DDT forms a myriad of over 45 metabolites and transformation products in biota and the environment, very few of which have been evaluated for environmental abundance or health effects. This reinforces the demand for high throughput and economical in vivo models for predictive toxicology screening, and the Japanese medaka is uniquely positioned to meet this need.

Cardiac and neurobehavioral impairments in three phylogenetically distant aquatic model organisms exposed to environmentally relevant concentrations of boscalid

Boscalid (2-Chloro-N-(4′-chlorobiphenyl-2-yl) nicotinamide), a pyridine carboxamide fungicide, is an inhibitor of the complex II of the respiration chain in fungal mitochondria. As boscalid is only moderately toxic for aquatic organisms (LC50 > 1–10 mg/L), current environmental levels of this compound in aquatic ecosystems, in the range of ng/L-μg/L, are considered safe for aquatic organisms. In this study, we have exposed zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) and Daphnia magna to a range of concentrations of boscalid (1–1000 μg/L) for 24 h, and the effects on heart rate (HR), basal locomotor activity (BLA), visual motor response (VMR), startle response (SR), and habituation (HB) to a series of vibrational or light stimuli have been evaluated. Moreover, changes in the profile of the main neurotransmitters have been determined. Boscalid altered HR in a concentration-dependent manner, leading to a positive or negative chronotropic effect in fish and D. magna, respectively. While boscalid decreased BLA and increased VMR in Daphnia, these behaviors were not altered in fish. For SR and HB, the response was more species- and concentration-specific, with Daphnia exhibiting the highest sensitivity. At the neurotransmission level, boscalid exposure decreased the levels of L-aspartic acid in fish larvae and increased the levels of dopaminergic metabolites in D. magna. Our study demonstrates that exposure to environmental levels of boscalid alters cardiac activity, impairs ecologically relevant behaviors, and leads to changes in different neurotransmitter systems in phylogenetically distinct vertebrate and invertebrate models. Thus, the results presented emphasize the need to review the current regulation of this fungicide.

Relative gene expression analysis of catalase in environmental RNA from Japanese medaka exposed to toxic chemicals

AbstractEnvironmental RNA (eRNA) has the potential as a non‐invasive tool for assessing the physiological status of macro‐organisms, yet the quantitative method for relative gene expression analysis is still underexplored. To bridge this gap, this study introduces a relative quantification method for eRNA, employing quantitative PCR (qPCR) to evaluate the expression of the antioxidant gene, catalase (cat), in the Japanese medaka Oryzias latipes exposed to two toxic chemicals for 96 h: chlorpyrifos (CPS) and carbamazepine. Our results showed that, in eRNA, genes frequently used as a reference for tissue or cells in conventional expression analysis correlated with each other. Also, one of them (elfa) exhibited less variability, showing its potential suitability as a reference gene in eRNA analyses. Additionally, cat expression levels in eRNA increased with increasing CPS concentrations, in a concentration‐response manner. These results suggest the promising potential of qPCR applications for eRNA in the monitoring of an organism's health and response to environmental changes. However, we observed disparities in gene expression levels of cat and beta‐actin (actb) between eRNA and whole fish body, indicating eRNA might have a biased origin. Further research is needed to uncover the origin of eRNA and determine the limitations and applicability domain of eRNA analysis.

Deciphering the origin of developmental stability: The role of intracellular expression variability in evolutionary conservation.

Progress in evolutionary developmental biology (evo-devo) has deepened our understanding of how intrinsic properties of embryogenesis, along with natural selection and population genetics, shape phenotypic diversity. A focal point of recent empirical and theoretical research is the idea that highly developmentally stable phenotypes are more conserved in evolution. Previously, we demonstrated that in Japanese medaka (Oryzias latipes), embryonic stages and genes with high stability, estimated through whole-embryo RNA-seq, are highly conserved in subsequent generations. However, the precise origin of the stability of gene expression levels evaluated at the whole-embryo level remained unclear. Such stability could be attributed to two distinct sources: stable intracellular expression levels or spatially stable expression patterns. Here we demonstrate that stability observed in whole-embryo RNA-seq can be attributed to stability at the cellular level (low variability in gene expression at the cellular levels). We quantified the intercellular variations in expression levels and spatial gene expression patterns for seven key genes involved in patterning dorsoventral and rostrocaudal regions during early development in medaka. We evaluated intracellular variability by counting transcripts and found its significant correlation with variation observed in whole-embryo RNA-seq data. Conversely, variation in spatial gene expression patterns, assessed through intraindividual left-right asymmetry, showed no correlation. Given the previously reported correlation between stability and conservation of expression levels throughout embryogenesis, our findings suggest a potential general trend: the stability or instability of developmental systems-and the consequent evolutionary diversity-may be primarily anchored in intrinsic fundamental elements such as the variability of intracellular states.

Single and mixture toxicity of cadmium and copper to swim bladder in early life stages of Japanese medaka (Oryzias latipes).

Toxicity observed in aquatic ecosystems often cannot be explained by the action of a single pollutant. Likewise, evaluation standards formulated by a single effect cannot truly reflect the environmental quality requirements. The study of mixtures is needed to provide environmental relevance and knowledge of combined toxicity. In this study, the embryos of Japanese medaka (Oryzias latipes) were treated with individual and binary mixture of copper (Cu) and cadmium (Cd) until 12days post-fertilization (dpf). Hatching, mortality, development, histology and gene expression were assessed. Our results showed that the highest concentration mixture of Cd (10mg/L) and Cu (1mg/L) affected survival, hatching time and hatching success. Occurrence of uninflated swim bladder was the highest (value) with exposure to 10mg/L Cd. Swim bladder was commonly over-inflated in a mixture (0.1mg/L Cd + 1.0mg/L Cu) exposure. Individuals exposed to the mixture (0.1 Cd + 1.0 Cu mg/L) showed up to a 7.69% increase in swim bladder area compared to the control group. The mixtures containing 0.1 or 10mg/L Cd, each with 1.0mg/L Cu resulted in significantly increased of Pbx1b expression, higher than any Cd or Cu alone (p < 0.01). In the co-exposure group (0.1/10 Cd + 1.0 Cu mg/L), Pbx1b expression was found at 12dpf but not 7dpf in controls. Higher concentrations of Cd may progressively reduce Pbx1b expression, potentially explaining why 75% of individuals in the 10mg/L Cd group failed to inflate their swim bladders. Additionally, the swim bladder proved to be a valuable bio-indicator for biological evaluation.

Effects of chronic diazepam exposure on the behaviors and oxidative stress homeostasis in the eyes and brains of female Japanese medaka

Diazepam (DZP) residue has been frequently detected in wastewater, surface water, and groundwater due to its extensive use over the decades. In this study, we exposed female Japanese medaka (Oryzias latipes) to environmentally relevant doses of DZP (800 and 8000 ng/L) for 4 weeks, aimed to investigate their behavioral responses and possible links with ocular and brain oxidative stress homeostasis. As a result, DZP exposure could significantly reduce swimming activity (800 ng/L) and anxiety (800 and 8000 ng/L), indicating a sedative effect on medaka. The DZP exposure also significantly increased the social interaction in medaka at 8000 ng/L. Furthermore, exposure to DZP could alter the ocular and brain oxidative stress homeostasis in medaka. The ocular CAT activities significantly increased in the 800 ng/L-DZP groups, and the brain SOD, CAT, GST and MDA levels also significantly increased in both DZP exposure groups. Correlation analysis revealed that the ocular and brain oxidative stress induced by DZP exposure might play an important role in their behavioral toxicity to medaka. Our findings highlight the necessity to clarify the exact link between DZP exposure-induced oxidative stress in the neural and sensor systems and its behavioral toxicity to better assess the risks on nontarget aquatic species.

Diazepam at environmentally relevant concentrations disturbed social interactions and brain neurotransmitters in adult Japanese medaka (Oryzias latipes)

Pollution by diazepam (DZP) is increasingly recognized as a major threat to aquatic organisms, but knowledge about its potential risk to fish is still limited. In this study, we exposed female and male Japanese medaka (Oryzias latipes) to environmentally relevant DZP (0.8 and 8 µg/L) for 28 days and investigated variation in their behavior (on days 7, 14, and 28) and brain neurotransmitter levels (on day 28). The results showed that DZP could be accumulated in the brain and gonads in Japanese medaka. When two fish of the same sex were placed in an aquarium, DZP exposure exhibited typical sedative effects on females (on day 7) and males (on days 7 and 14). However, these sedative effects on both sexes were no longer present after 28 days of exposure. Exposure to DZP induced sex-specific impacts on the social interactions of medaka on days 7, 14, and 28 of exposure in a time-dependent manner. When both sexes were placed into an aquarium in a ratio of 1:1, DZP could significantly alter their locomotor activity and social interaction on days 14 and 28 of the exposure. After 28 days of exposure, DZP significantly altered the levels of several neurotransmitters in the brain of medaka, also in sex-specific manners. The alterations in dopamine and serotonin levels exhibited significant correlations with the increased social interaction between females. At the same time, that of γ-aminobutyric acid significantly correlated to the decreased social interaction between males. Our findings suggest that chronic exposure to DZP, even at environmentally relevant concentrations, can accumulate in the brains and gonads of fish, and alter their behaviors by mediating brain neurotransmitter levels, which may further disturb their reproduction and population dynamics.

Japanese medaka (Oryzias latipes) exposed via maternal transfer to the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), experience decreased fecundity and impaired oocyte maturation

Early life-stage exposure of fishes to endocrine disrupting chemicals can induce reproductive impairment at sexual maturity. Previously, we demonstrated decreased fecundity of Japanese medaka (Oryzias latipes) exposed via maternal transfer to the novel brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO). However, that study failed to identify the causative mechanism. In other studies we have shown that decreased fecundity of adult fish exposed to dietary TBCO is likely due to impaired oocyte maturation. The goal of the present study was to determine if impaired oocyte maturation is responsible for decreased fecundity of Japanese medaka exposed as embryos to TBCO, via maternal transfer. Sexually mature fish (F0) were fed either a control diet or a low (74.7 μg/g) or high (663 μg/g) diet containing TBCO for 21 days. Eggs (F1) were collected during the final week of exposure and reared to sexual maturity at which point fecundity was assessed using a 21-day reproduction assay. Upon termination of the assay, an ex vivo oocyte maturation assay was used to determine whether maturation inducing hormone (MIH) stimulated oocyte maturation was impaired. Additionally, concentrations of 17β -estradiol (E2) in blood plasma and expression of genes involved in vitellogenesis and oocyte maturation were quantified. The F1 generation females reared from the low or high F0 treatments experienced a 26.0 % and 56.8 % decrease in cumulative fecundity, respectively. Ex vivo MIH stimulated oocyte maturation from the low and high TBCO treatments were decreased by 23.4 % and 20.0 % respectively. There was no significant effect on concentrations of E2. Transcript abundance of vtgI was significantly decreased in a concentration dependent manner. Transcript abundance of mPRα, pgrmc1, pgrmc2, and igf3 were decreased but effects were not statistically significant. Overall, results suggest that impaired oocyte maturation causes decreased fecundity of Japanese medaka exposed to maternally deposited TBCO.

Neuropeptide Y receptor Y2 (npy2r) deficiency reduces anxiety and increases food intake in Japanese medaka (Oryzias latipes).

Neuropeptide Y receptor Y2 (npy2r) is an important receptor gene involved in anxiety and feeding regulation in mammals. Since NPY receptors have different receptor gene deletions in mammals and teleost fish, it is not clear whether npy2r has the similar function in fish as in mammals. In this study, we used the CRISPR/Cas9 system to establish npy2r-deficient medaka (Oryzias latipes). Unexpectedly, the deletion of npy2r resulted in the npy2r +/- medaka were all-male, therefore, npy2r homozygous mutant lines could not be established. The deletion of npy2r increased the food intake in medaka, and the expression levels of appetite stimulating genes (agrp, npy) increased significantly, while the expression levels of anorexia factors (cck, pomc) decreased significantly. Moreover, the absence of npy2r significantly increased the total length and body weight of medaka. The mirror test and open field test showed that npy2r +/- medaka improved sociability and reduced anxiety-like behavior, qRT-PCR analysis showed that the expression levels of anxiety related genes (th1, th2, gr1, gr2, and mr) in npy2r +/- medaka were significantly decreased. So far, this is the first npy2r gene knockout model established in fish and demonstrates that npy2r plays an important role in the regulation of reproduction, feeding and anxiety in fish.

Influence of phthalate and non-phthalate plasticizers on reproductive endocrine system-related gene expression profiles in Japanese medaka (Oryzias latipes)

Plasticizers containing phthalates have the potential to alter endocrine function in vertebrates. While non-phthalate plasticizers were previously considered to be environmentally friendly and safe, our research team discovered that bis-(2-ethylhexyl) adipate (DEHA) and acetyl tributyl citrate (ATBC) disrupt thyroid hormones in Japanese medaka (Oryzias latipes). We assessed reproductive- and estrogen-responsive gene expression patterns in Japanese medaka to determine whether the phthalate plasticizers bis-(2-ethylhexyl) phthalate (DEHP, positive control) and the non-phthalate plasticizers DEHA and ATBC disrupt endocrine signaling. The results showed that the levels of choriogenin H (chgH) and vitellogenin (vtg) genes increased after exposure to DEHP and ATBC, suggesting that these plasticizers may have estrogenic activity. Exposure to DEHP and DEHA resulted in the upregulation of kisspeptin (kiss), gonadotropin-releasing hormone (gnrh), and follicle-stimulating hormone beta (fshβ) genes, suggesting that these plasticizers may interfere with reproductive function. To the best of our knowledge, this is the first study to demonstrate that the non-phthalate plasticizers DEHA and ATBC can disrupt reproduction-related hormonal activity in fish.

Sarcolipin (sln) and Sarcoplasmic Reticulum calcium ATPase pump (serca1) expression increase in Japanese medaka (Oryzias latipes) skeletal muscle tissue following cold challenge

Endothermy is the process by which organisms maintain a constant body temperature despite dynamic environmental temperatures. There are two mechanisms organisms use to elevate body temperature: shivering thermogenesis (ST) and non-shivering thermogenesis (NST). Skeletal muscle NST is achieved through a futile Ca2+ cycling of sarcoplasmic reticulum Ca2+ ATPase (Serca1) in the presence of sarcolipin (Sln). Here we subjected Japanese medaka to a cold challenge to examine the expression of sln and serca1 transcripts from slow-twitch red and fast-twitch white muscle as environmental temperature decreased. We show a significant increase in relative sln and serca1 transcript expression in skeletal muscle tissues of cold-challenged Japanese medaka. The elevated transcripts support the role of Sln as a component of NST and support previous literature with the increase in serca1. To date, this is the first cold challenge on an ectothermic fish investigating sln transcripts. The ability of medaka to respond to a cold challenge with an increase in key calcium cycling components, specifically the calcium pump and sarcolipin suggest that teleost fish share a conserved transcriptional program in response to cold stimuli with fish species that possess the requisite anatomical adaptations to conserve metabolic heat.

Effects of Chronic Roundup Exposure on Medaka Larvae.

The use of glyphosate-based herbicides is increasing yearly to keep up with the growing demands of the agriculture world. Although glyphosate-based herbicides target the enzymatic pathway in plants, the effects on the endocrine systems of vertebrate organisms, mainly fish, are widely unknown. Many studies with glyphosate used high-exposure concentrations (mg/L), and the effect of environmentally relevant or lower concentrations has not been clearly understood. Therefore, the present study examined the effects of very low, environmentally relevant, and high concentrations of glyphosate exposure on embryo development and the thyroid system of Japanese medaka (Oryzias latipes). The Hd-rR medaka embryos were exposed to Roundup containing 0.05, 0.5, 5, 10, and 20 mg/L glyphosate (glyphosate acid equivalent) from the 8 h post-fertilization stage through the 14-day post-fertilization stage. Phenotypes observed include delayed hatching, increased developmental deformities, abnormal growth, and embryo mortality. The lowest concentration of glyphosate (0.05 mg/L) and the highest concentration (20 mg/L) induced similar phenotypes in embryos and fry. A significant decrease in mRNA levels for acetylcholinesterase (ache) and thyroid hormone receptor alpha (thrα) was found in the fry exposed to 0.05 mg/L and 20 mg/L glyphosate. The present results demonstrated that exposure to glyphosate formulation, at a concentration of 0.05 mg/L, can affect the early development of medaka larvae and the thyroid pathway, suggesting a link between thyroid functional changes and developmental alteration; they also showed that glyphosate can be toxic to fish at this concentration.

Characterization of Fibrodysplasia Ossificans Progessiva relevant Acvr1/Acvr2 Activin receptors in medaka (Oryzias latipes).

Activin and Bone Morphogenetic Protein (BMP) signaling plays crucial roles in vertebrate organ formation, including osteo- and angiogenesis, and tissue homeostasis, such as neuronal maintenance. Activin and BMP signaling needs to be precisely controlled by restricted expression of shared receptors, stoichiometric composition of receptor-complexes and presence of regulatory proteins. A R206H mutation in the human (hs) BMP type I receptor hsACVR1, on the other hand, leads to excessive phosphorylation of Sons of mothers against decapentaplegic (SMAD) 1/5/8. This in turn causes increased inflammation and heterotopic ossification in soft tissues of patients suffering from Fibrodysplasia Ossificans Progressiva (FOP). Several animal models have been established to understand the spontaneous and progressive nature of FOP, but often have inherent limitations. The Japanese medaka (Oryzias latipes, ola) has recently emerged as popular model for bone research. To assess whether medaka is suitable as a potential FOP animal model, we determined the expression of Activin receptor type I (ACVR1) orthologs olaAcvr1 and olaAcvr1l with that of Activin type II receptors olaAcvr2ab, olaAcvr2ba and olaAcvr2bb in embryonic and adult medaka tissues by in situ hybridization. Further, we showed that Activin A binding properties are conserved in olaAcvr2, as are the mechanistic features in the GS-Box of both olaAcvr1 and olaAcvr1l. This consequently leads to FOP-typical elevated SMAD signaling when the medaka type I receptors carry the R206H equivalent FOP mutation. Together, this study therefore provides experimental groundwork needed to establish a unique medaka model to investigate mechanisms underlying FOP.