Fish husbandry and care
AB wild type fish were utilized for this study. Zebrafish were housed in a 700 L recirculating zebrafish aquarium system (Aquatic Enterprises, Seattle, WA) regulated by a Profilux 3 Outdoor module that regulated salinity, pH, and temperature (GHL International, Kaiserslautern, Germany) 24 h a day. The facility was illuminated on a 12 h light/ 12 h dark cycle. Zebrafish were fed once a day with hatched brine shrimp (Brine Shrimp Direct, Ogden, UT) and once a day with Gemma micro 300 (Skretting, Westbrook, ME).
Isolation box experiments
An isolation box was constructed from ¾” plywood and placed over the breeding tanks. The box was designed to accommodate 8 mating chambers total and hold fluorescent light bulbs on its ceiling to create specific lighting conditions, as well as exclude lighting from the surrounding area. The box was divided into 4 equal chambers so that 2 tanks were exposed to red light, 2 tanks were exposed to blue light, 2 tanks were exposed to green light, and 2 tanks were exposed to soft white light simultaneously. Inside the top of the box was a standard E26 light bulb socket wired to a timer. Treatment lights were fluorescent 13 W bulbs in red (BPESL13T/R, Feit Electric), blue (T3 Twister, Phillips Lighting), green (BPESL13T/G, Feit Electric), and soft white (13 W, Feit Electric) coloring. Facility lights were used as a control (see Fig. 1B for wavelengths). The isolation box lighting was synchronized with the facility lights, which allowed fish to maintain their 12 h light/ 12 h dark cycle. Light wavelengths for the fluorescent bulbs were assessed inside the isolation box with a spectrometer (USB2000, Ocean Optics, Dunedin, FL), and light intensity was assessed in the same location with a light meter (401027, Extech Instruments, Nashua, NH).
Experimental design: breeding tank setup
Three 9 L tanks (Tanks A, B, and C) housing 30 6-month-old wt zebrafish each (10 males and 20 females) were utilized. Every week, 10 total crosses (2 females to 1 male) were set up in 10 separate DuraCross zebrafish breeding tanks (Laboratory Products Sales, Rochester, NY). All breeding tank inserts were inclined to create a depth gradient within the chamber. Every Monday, all the fish in Tank A were mated to each other. This was repeated for Tank B on Wednesday and Tank C on Friday. In this way, every week each tank of fish was mated. These matings were subjected to our modulated lighting conditions. This pairing continued for 16 weeks.
Experimental design: lighting
Treatment began at the onset of the dark-cycle (21:00 h). At this time, the isolation box was placed over 8 breeding chambers and left there overnight. The next morning at the transition from the dark-light cycle (09:00 h), the timer illuminated treatment lights inside the box. This treatment lasted for 4 h. At the end of the treatment, embryos were collected, placed in embryo media, and counted the same day. A control treatment (with room lights) was carried out at the same time; 2 mating chambers were left on the counter next to the isolation chamber, exposing the fish to the facility lighting for 4 h. After completion of treatment, individual fish were put back into their designated housing tanks. Room temperature, humidity, water temperature, water conductivity, and water pH was also recorded every week. The embryos were placed in a 28°C incubator and counted later that afternoon. Viability of the embryos was assessed the next day.
iSpawn-S experiment
An iSpawn-S system (Techniplast, Westchester, PA) with minor modification was utilized for breeding; LED light bulbs were placed into the top of the plastic lid. Large dark bags were placed over the iSpawn-S to exclude lighting from the surrounding area. Treatment lights were adjustable LED deck lights in red, blue, and green (Paradise lighting, Model GL34001SS6). Facility lights were used as a control (see Fig. 1D for wavelengths). The lighting was synchronized with the facility lights, which allowed fish to maintain their 12 h light/ 12 h dark cycle. Light wavelengths for LED lights were assessed inside the iSpawn-S with a spectrometer (USB2000, Ocean Optics, Dunedin, FL), and light intensity was assessed in the same location with a light meter (401027, Extech Instruments, Nashua, NH).
Experimental design: breeding setup
Two 9 L tanks (Tanks D and E) housing 8 6-month-old wt zebrafish each (4 males and 4 females) were utilized. Every week 2 females were removed from Tank D and 2 females were removed from Tank E, and placed in the bottom section of the iSpawn-S. 2 males were then removed from Tank D and Tank E, and placed in the top section of the iSpawn-S. In this way, there were 4 females and 4 males per breeding. Every week each tank of fish was mated. These matings were subjected to our modulated lighting conditions. This pairing continued for 20 weeks.
Experimental design: lighting
Treatment began at the onset of the dark cycle (21:00 h). At this time, bags were placed over the iSpawn-S and left there for overnight. The next morning at the transition from the dark light cycle (09:00 h), the timer illuminated treatment lights inside the device, and the separator between males and females was removed. This treatment lasted for 4 h. At the end of the treatment, embryos were collected, placed in embryo media, and counted the same day. Control treatments (with room lights) were also performed. After mating, individual fish were put back into their designated housing tanks. Room temperature, humidity, water temperature, water conductivity, and water pH was also recorded every week. The embryos were placed in a 28°C incubator and counted later that afternoon. Viability of the embryos was assessed the next day.