Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

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

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

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

Subcellular redistribution and sequential recruitment of macromolecular components during SGIV assembly

Virus infection consists of entry, synthesis of macromolecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus (SGIV) assembly in the medaka haploid embryonic stem (ES) cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluorescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site (VAS). Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell. Taken together, VP088 is an excellent marker for visualizing the SGIV infection process. Our results provide new insight into macromolecular component recruitment and SGIV assembly.

Aquatic Toxicity Assessment of Phosphate Compounds

OBJECTIVES: Tricalcium phosphate and calcium hydrogenorthophosphate are high production volume chemicals, mainly used as foodstuff additives, pharmaceuticals, lubricants, synthetic resin, and disinfectants. Phosphate has the potential to cause increased algal growth leading to eutrophication in the aquatic environment. However, there is no adequate information available on risk assessment or acute and chronic toxicity. The aim of this research is to evaluate the toxic potential of phosphate compounds in the aquatic environment. METHODS: An aquatic toxicity test of phosphate was conducted, and its physico-chemical properties were obtained from a database recommended in the Organization for Economic Cooperation and Development (OECD) guidance manual. An ecotoxicity test using fish, Daphnia, and algae was conducted by the good laboratory practice facility according to the OECD TG guidelines for testing of chemicals, to secure reliable data. RESULTS: The results of the ecotoxicity tests of tricalcium phosphate and calcium hydrogenorthophosphate are as follows: In an acute toxicity test with Oryzias latipes, 96 hr 50% lethal concentration (LC50) was >100 (measured:>2.14) mg/L and >100 (measured: >13.5) mg/L, respectively. In the Daphnia test, 48 hr 50% effective concentration (EC50) was >100 (measured: >5.35) mg/L and >100 (measured: >2.9) mg/L, respectively. In a growth inhibition test with Pseudokirchneriella subcapitata, 72 hr EC50 was >100 (measured: >1.56) mg/L and >100 (measured: >4.4) mg/L, respectively. CONCLUSIONS: Based on the results of the ecotoxicity test of phosphate using fish, Daphnia, and algae, L(E)C50 was above 100 mg/L (nominal), indicating no toxicity. In general, the total phosphorus concentration including phosphate in rivers and lakes reaches levels of several ppm, suggesting that phosphate has no toxic effects. However, excessive inflow of phosphate into aquatic ecosystems has the potential to cause eutrophication due to algal growth.

Medaka Fish Parkinson's Disease Model

The teleost fish has been widely used in creating neurodegenerative models. Here we describe the teleost medaka fish Parkinson's disease (PD) models we developed using toxin treatment and genetic engineering. 1-Methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors and tunicamycin treatment in our model fish replicated some salient features of PD: selective dopamine cell loss and reduced spontaneous movement with the last three toxins producing inclusion bodies ubiquitously in the brain. Despite the ubiquitous distribution of the inclusion bodies, the middle diencephalic dopaminergic neurons were particularly vulnerable to these toxins, supporting the idea that this dopamine cluster is similar to the human substantia nigra. PTEN-induced putative kinase 1 (PINK1) homozygous mutants also showed reduced spontaneous swimming movements. These data indicate that medaka fish can serve as a new model animal of PD. In this review we summarize our previous data and discuss future prospects.

Growth inhibition in Japanese medaka (Oryzias latipes) fish exposed to tetrachloroethylene.

A recent study in our laboratory has demonstrated that tetrachloroethylene (TCE) is acutely toxic to Japanese medaka (Oryzias latipes) larvae with a 96 hr-LC50 of 18 (17-19) mg/mL (Spencer et al., 2002). In the present study we hypothesize that TCE exposure induces a developmental effect in Japanese medaka. Growth and age specific sensitivity of Japanese medaka larvae were studied with four age groups (7, 14, 21 and 28 days old) to determine tetrachloroethylene effects on these parameters. The medaka larvae were exposed for 96 hours in a single concentration (10 mg/mL) of TCE. The toxic endpoints evaluated were larvae weight, length, water content and protein concentration. The study revealed that exposure of medaka larvae to this sub-acute concentration of TCE significantly reduced length and weight in the treated group. The difference in growth between control and treated groups was more obvious in age versus length, than in age versus weight. The dry weight-fresh weight ratio (dw/fw) was shown to be higher in the control group. Water content in TCE-treated medaka was higher than in the control group, and younger fry had more water content than older ones. A higher protein concentration was also observed in TCE-treated medaka compared to the control group. These results indicate that TCE has a profound effect on the growth and development of Japanese medaka larvae.

Developmental arrest in Japanese medaka (Oryzias latipes) embryos exposed to sublethal concentrations of atrazine and arsenic trioxide.

Embryos of the Japanese medaka (Oryzias latipes) were exposed to serial concentrations of atrazine (0, 25, 50, and 100 ppm) and arsenic trioxide (0, 0.025, 0.05, 0.1 ppm) until hatching. Stasis of circulation, blood islands, titanic convulsions, tube heart and mortality were observed in atrazine-treated embryos. Each endpoint exhibited a concentration-response relationship. Only 4% of the embryos hatched in the 25 ppm, and none in the 50 and 100 ppm, probably due to cell death attributed to the embryos' inability to break from the chorion. With arsenic exposure, hatching was inversely correlated to chemical concentration: 86%, 75% and 54% for 0.025, 0.05 and 0.1 ppm, respectively. Hatching periods were also reduced from 7-13 days in controls to 7-11 days in arsenic-treated embryos. This observation was more pronounced with the 0.05 ppm concentration, showing a reduction of about 4 days. Despite this shortage in hatching time, there were no observable morphological abnormalities, as seen with atrazine. The ecological significance of these findings and implications for the development of sublethal toxicity tests using Japanese medaka embryos are important.