All fish are maintained in the animal facility fish of the Leibniz Institute on Aging – Fritz Lipmann Institute Jena consistent with the German Animal Welfare Law.

Husbandry conditions include a water temperature of 27 °C ± 1 °C, conductivity of approximately 0.8 mS, and a 12:12-h light:dark cycle. Water quality parameters, including pH, temperature, and conductivity, are monitored and recorded continuously. Nitrate and nitrite are measured daily. Group-housed fish are supplied with certified contaminant-screened blue polycarbonate igloos, retreats, huts, and tents as enrichment. Permanent breeding groups consist of one male and 4 females and are provided with a sand bowl for oviposition. Embryos are collected weekly on coconut fiber substrate and incubated at 23 °C in dedicated incubators.

Freshly hatched larvae are fed twice daily with live first instar brine shrimp nauplii (Artemia sp.) and then weaned at the age of 4 to 6 wk onto live bloodworms (Chironomidae) once a day. Fish are tracked on an individual basis by using the commercially available database Tick@lab. Fish are visually inspected daily by animal technicians with reference to the killifish health score sheet (Naumann et al., 2023). Health scores of fish exhibiting clinical signs were documented in Tick@lab.

Health monitoring program: The routine quarterly killifish colony health surveillance program incorporates both molecular methods using whole fish (sentinels) and environmental samples (sump) for molecular analysis and whole fish for histopathology (sentinels). Adult postfiltration sentinel N. furzeri are placed in sentinel tanks in each rack for a minimum exposure period of 3 mo. Sentinels are euthanized by hypothermal shock (rapid chilling) or overdose with buffered tricaine methanesulphonate (MS-222, 1g/L) and submitted to IDEXX BioAnalytics (Columbia, MO) for individual evaluation by histopathology, pooled into groups of 5 for evaluation by real-time PCR, or analyzed individually in-house by conventional PCR for microsporidia. Each health monitoring round samples 2.7% of the entire fish colony. In addition, biofilter samples are collected from the sump of each rack as environmental samples and are processed individually by real-time PCR.