Anadromous salmon migrate between fresh water (FW) and seawater (SW) during their life cycle, which most likely is driven by changes in salinity preference. There have been some studies examining salinity preference in juveniles during downstream migration, but no study has yet been reported in homing adults. In this study, we established an aquarium system to evaluate salinity preference that is usable not only for juveniles but for adult fish. The aquarium consists of three areas of different salinities, a FW area, a SW area and a brackish water (BW) area, among which fish can voluntarily move. By modifying the flow rate of FW/SW and other parameters, we could maintain the salinity of the FW area at 20 ppt irrespective of the depth, but the salinity varies considerably by depth between the surface and bottom layer in the BW area. Two aquaria with the same system were prepared side by side, an acclimation aquarium that allowed fish to learn the system before the experiment and an experimental aquarium that was insulated from the environment and observed by a video system. Using this aquarium system, we found that cultured rainbow trout, Oncorhynchus mykiss, of ca. 200g preferred areas in the order of BW≥FW>>SW. To further validate this system, we injected various hormones that have been implicated in osmoregulation/smoltification such as angiotensin II (Ang II), cortisol, thyroid hormone (T3), arginine vasotocin (AVT) and prolactin into the third brain ventricle of the trout and observed their preference behavior. There was a tendency toward high salinity preference after injection of hormones that promote SW acclimation and/or smoltification (Ang II, cortisol T3 and AVT), but fish tended to prefer a low salinity area after injection of prolactin that promotes FW adaptation. These results suggest that the newly established aquarium system can be used to evaluate salinity preference in salmonids and will contribute to future studies in identidying key factors that motivate downstream migration of juveniles and upstream migration of mature chum salmon, O. keta, homing to natal rivers in the Tohoku area of Japan
