The genus Oryzias containing freshwater (FW) and seawater (SW) species is a potential model for studying mechanisms of osmotic adaptation. In this study, we compared SW adaptability of four Oryzias species, O. javanicus, O. dancena, O. latipes and O. marmoratus inhabiting different osmotic environments. SW adaptability was evaluated at several stages of the lifecycle: (i) survival rates of adult fish after transfer from FW to 50%SW or SW, (ii) spawning ability in FW and SW, (iii) fertilization rates in FW and SW, and (iv) hatching rates in FW, 50%SW and SW. Results obtained agreed with the natural habitat of each species: O. javanicus, which inhabits SW or brackish water (BW), is fully adaptable to both SW and FW at all the stages examined. The BW species O. dancena also revealed high SW adaptability except for the hatching rate. O. marmoratus, confined in FW, exhibited low SW adaptability at all stages examined while O. latipes, another FW species, was adaptable to SW at most stages examined. Based on these results, the role of SW adaptability to the distribution area of each species is discussed.
The genus Oryzias consists of more than 14 species including medaka (O. latipes), a species well known as a laboratory animal (Naruse et al., 1993; Naruse, 1996; Roberts, 1998). Each species of this genus reveals a specific pattern of geographic distribution; some species are endemic to freshwater (FW) lakes in an Indonesian Island, while others are distributed widely in FW, brackish water (BW) and seawater (SW) area of Asia (Naruse et al., 1993; Naruse, 1996; Roberts, 1998). Since such distribution patterns have hardly been altered by human activities because of low commercial value, Oryzias species offer interesting examples for studies of species diversity, evolution and ecology.
The genus Oryzias is also a potential model for studies of adaptation to hyper- and hypoosmotic environments because diverse osmotic adaptability among species is expected. In this paper, we compared the SW adaptability of four Oryzias species, O. javanicus, O. dancena, O. latipes and O. marmoratus inhabiting different osmotic envronments. O. javanicus is found in SW and BW of Thailand, the Malaysian Peninsula and Indonesian Islands (Iwamatsu et al., 1982; Naruse, 1996; Roberts, 1998). O. dancena (also known as O. melastigma) is widely distributed in BW and FW ranging from Pakistan and India to Burma and Thailand (Naruse, 1996; Roberts, 1998). O. latipes is distributed widely in FW of Japan, Korea and China (Naruse et al., 1993; Naruse, 1996) although it is sometimes found in BW (Miyamoto et al., 1986). O. marmoratus is endemic to Lake Towuti, a highland FW lake of Sulawesi Island, where Adrianichthid fish including Oryzias are highly diversified (Naruse et al., 1993; Naruse, 1996). The results showed that adaptability to SW at different life stages correlated with distribution area in their natural habitats.
MATERIALS AND METHODS
All experiments were carried out according to the guideline for animal experiments, Faculty of Science, University of Tokyo. O. javanicus sampled at Penan, Malaysia was obtained from Dr. J. Koyama of Kagoshima University, O. dancena was from Dr. K. Naruse of University of Tokyo, HNI strain of O. latipes was from Drs. A. Shima and A. Shimada of University of Tokyo, and O. marmoratus was from Drs. M. Sakaizumi of Niigata University and K. Naruse of University of Tokyo. All experiments were performed at 26°C. Aged tap water and natural water of North Pacific Ocean (approximately 35‰) were used as FW and SW, respectively. O. javanicus was bred and maintained in SW and other three species were in FW.
Survival rate of Adult fish
For each species, 24 individuals acclimated to FW or SW for 2 wks were prepared. They are divided into 3 groups of 8 individuals and transferred directly into small plastic aquaria filled with 3L of FW, 50%SW and SW, respectively. The survival was monitored every 1 hr for 25 hr. The death of fish was defined as the stopped movement of the operculum. In O. latipes, an additional experiment was performed: 8 individuals that have been reared only in FW were transferred to SW after 24 hr acclimation to 50% SW and the survival was monitored everyday for 30 days.
Observation of spawning
Four adult male and 4 adult female of each species were maintained in a 18L aquarium filled with FW or SW under the controlled photoperiod of 14 hr light and 10 hr dark. Spawning was watched everyday for 1 hr from the start of the light period during which spawning always occurred.
Spawned eggs were collected from the abdomen of females of each species approximately 5 hr after the start of light period. Eggs in which cleavage progressed normally to 16–128 cell stage (Stages 6–8 of Iwamatsu, 1994) or more advanced stages were counted as fertilized. In our preliminary experiment, it was found that most unfertilized eggs have unbroken cortical alveoli just after spawning. Although some of them are sometimes found activated by unknown stimuli a few hours after spawning, they are distinguishable from fertilized eggs because they do not reveal regular patterns of cleavage.
The testis was isolated from 3 adult males of each species that had maintained in FW for more than 30 days after decapitation. It was divided into 2 pieces using forceps and each piece was put into a drop of FW and SW on a slide glass, respectively. Sperm cells were suspended by tearing the piece of the testis using forceps and the rate of motile sperm cells was estimated immediately after suspension according to the standard of Suquet et al. (1992) under the microscope (BX-51, Olympus, Tokyo).
Fertilized eggs obtained in FW or SW were transferred to FW, 50% SW and SW about 5 hr after spawning. Eggs were incubated separately using 96-well culture plates at 26°C. Embryos that died before hatching and those that successfully hatched were counted everyday. Embryos in which collapse of the morphology or stop of the heartbeat was observed were regarded as dead.
To examine hatching ability separately from the difference in development, FW-spawned fertilized eggs of O. javanicus, O. dancena and O. latipes were collected about 5 hr after spawning and incubated in SW until the hatching of the first individual. After the hatching of the first fish, other embryos were divided into two groups, transferred into newly prepared FW and SW, respectively, and incubated at 28°C. Hatching was checked every 30–120 min for 24 hr.
Survival of adult fish after direct transfer
All O. javanicus and O. dancena survived the transfer to 50% SW and SW (Fig. 1). In contrast, all O. marmoratus and O. latipes transferred to SW died within 2 hr and 9.5 hr, K. Inoue and Y. Takei respectively. The average time of survival in SW was, 1.6±0.2 hr in O. marmoratus and 4.3±0.8 hr in O. latipes, respectively, which was significantly different from each other (p<0.01). O. marmoratus was not adaptable even to 50% SW; all fish died within 25 hr (19.3±1.7 hr in average) after transfer to 50% SW. On the other hand, O. latipes survived in 50% SW for more than 25 hr (Fig. 1) and no fish died after elongated exposure to 50% SW for up to 48 hr (data not shown). It was also found that prior acclimation of O. latipes in 50% SW for 24 hr allowed them to survive in SW for more than 30 days. SW-acclimated O. javanicus, O. dancena and O. latipes could survive the transfer back to FW.