Temperature–Salinity Dependence of the Mitotic Interval (0) for Chromosomal Manipulation in Marine Medaka, Oryzias dancena

Eggs of the marine medaka, Oryzias dancena were collected and fertilized to
observe the temperature-salinity-related cleavage rates and mitotic intervals (0). We
investigated the relationship between 0 and five different water temperatures (18, 22,
26, 30, and 34°C) and four different salinities (0, 10, 20, and 30 ppt NaCl). As the water
temperature increased, the slope of the first cleavage frequency increased with elapsed
time after fertilization, and approximately 30% of fertilized eggs reached the first
cleavage frequency at every 15-minute intervals. However, the slope of the first cleavage
frequency did not differ significantly between 0 ppt and the other salinities (10, 20, or
30 ppt). At higher water temperatures, the eggs developed more rapidly, but no other
developmental process was affected. At higher salinity, the hatching rates of the eggs
decreased, and the hatching times were delayed. There was a strong negative correlation
between 0 and water temperature as shown in this equation (Y = –1.137X + 75.47, R2 =
0.977, where Y is 0 and X is water temperature). At a constant water temperature, 0
did not differ significantly in 0, 10, 20, and 30 ppt NaCl.

applied [8][9][10]14,15]. The genetic material of a gynogenetic haploid organism can be doubled by controlling the second meiotic division and the first cleavage. The second meiotic division can be controlled by water temperature, hydrostatic pressure, or chemical treatment, which can also be used to induce triploidy [15,16]. The first cleavage can be controlled by heat shock or hypostatic pressure, which are also used individually or sequentially to induce tetraploidy [17][18][19][20].
The effectively controlled release of the second ootid and the first cleavage are dependent on the type, intensity, and duration of the treatment [21][22][23]. The method of controlling the first cleavage as a means of chromosomal engineering can also enhance shortterm production in aquaculture [14]. It also has an application in the induction of tetraploidy, mitotic gynogenetic diploidy, or androgenetic diploidy using chromosomal engineering. To effectively control the first cleavage, an understanding of its temperature dependence is essential [14,24]. However, determination of mitotic interval is necessary for producing triploid and tetraploid.
The Dettlaff unit (mitotic interval,  0 ) is the duration (in minute) of one mitotic cycle during the early synchronous embryonic cleavage or the interval between two consecutive cell divisions [18,20,25,26]. When measured over a range of water temperatures, the relationship between  0 and water temperature, as determined by regression analysis, can showthe developmental events that are influenced by temperature within a species and between species with a similar spawning biology [18,20,27] [26,[28][29][30][31][32][33][34][35][36]. Because the marine medaka has a wide range of salinity tolerance, we identified the egg development achieved through artificial fertilization, and also assessed the temperature-salinity dependence of  0 and the embryo cleavage rates to establish the most efficient procedures for chromosome manipulation in the marine medaka.

Materials and Methods
The experimental group of diploid marine medaka, Oryzias dancena used in this study was reared according to the methods of the previous study [37]. Fish with a standard length of over 25 mm were used in this experiment; 35 males and 15 females were placed in each of two aquariums, and 2,000 fertilized eggs were collected immediately by net. The fertilized eggs of the experimental group (n = 100) were reared in a 100 L glass aquarium. To assess the temperature-salinity dependence of the first cleavage and  0 , the water temperature was maintained in temperature-controlled water baths at 18, 22, 26, 30, or 34°C, and each experimental group was subjected to one of four salinities, 0, 10, 20, or 30 ppt NaCl. The water salinity of each group was regulated to remain constant throughout the experiment. Samples were generally taken at 5-minute intervals and fixed with 5% neutral formalin solution (50 mL formalin, 3.25 g Na 2 HPO 4 ·12H 2 O, 2.25 g KH 2 PO 4 , 950 mL distilled water) at 4°C before observation. We measured the diameter of 10 fertilized eggs at a magnification of 50 under an optical microscope (Axiostar plus, Zeiss, Germany) equipped with a microscope camera (Axiocam MR, Zeiss). A specific developmental stage was considered to be reached when approximately 80% of the developing embryos were at that stage. In this study, all experiments were performed in triplicate.
The time of the appearance of the first cleavage furrow was recorded and was deemed to be the start of the subsequent cell division. The times at which approximately 10% of the developing embryos reached the two-cell ( I ; Figure 1a) and eight-cell ( III ; Figure 1b) stages were recorded. The value of 10% was selected according to the recommendation of the previous study [38]. The mean  0 value was calculated as  0 = ( III - I )/2. The relationship between the mean  0 and the water temperature was examined by simple linear regression line. One-way and two-way ANOVA analysis were applied to determine water temperature and salinity effects. Duncun's multiple range test was applied when significant difference was observed.

Result
The fertilization, hatching rates, and hatching times of the marine medaka, Oryzias dancena eggs are shown in Table 1. The fertilization rates of the groups were not significantly different, and the hatching rates of each group did not differ significantly as the water temperature increased. With increasing water salinity, the hatching rates decreased at each specific water temperature, but the hatching rates of each group did not differ significantly at a specific salinity respectively. With increasing water salinity, the hatching time of each group was delayed in each specific water temperature and its time was delayed as the water temperature decreased in each specific salinity respectively. As water temperature increased, the two-cell stage and the eight-cell stage were reached earlier in a specific salinity. However, the times taken to reach the two-cell stage and eight-cell stage did not differ across different salinities at the same water temperature (Table 1).

Discussion
The previous study has reported the embryogenesis and early ontogenesis of the marine medaka, Oryzias dancena [2][3]. As they mentioned, the marine medaka eggs reaches the two-cell stage 90 minutes after fertilization at 26°C and 1 ppt NaCl and reaches the eight-cell stage 3 hrs after fertilization under these conditions.
Although the experimental salinities used in our study differed from that of the previous study, the times required to reach the two-cell stage and eight-cell stage at 26°C and 0 ppt NaCl in our study were similar to the previous study at 26°C and 1 ppt NaCl [2]. Another previous study also reported the embryonic development and early viability of the marine medaka under different salinity conditions [38]. They reported that the hatching rates of their eggs decreased at higher salinities, and the hatching rate of the eggs was highest at 0 ppt NaCl among the salinities tested [38]. At higher salinities, the hatching times of the eggs were delayed, and its hatching time at 0 ppt NaCl was earliest among the experimental groups. In the salinity range of 0 ppt to 30 ppt NaCl, the hatching rates and hatching times in our study were similar to those of the previous study [38].
We also attempted to determine the water temperature-salinity dependence of the cleavage rates and  0 and found that the marine medaka eggs underwent cleavage within a temperature range of 18-34°C and a salinity range of 0-30 ppt NaCl. We found that the marine medaka eggs showed faster development with increasing water temperatures and water salinity, whereas  0 decreased with increasing water temperatures, which indicates a strong negative correlation between  0 and water temperature. Although  0 and hatching time after fertilization differed greatly from those of other  [18,20,25,[32][33][34][35][36]39].
The relationship between  0 and water temperature in fish is typically curvilinear, when the temperatures are within the range in which the fish species naturally spawn and develop [18,20,25,[32][33][34][35]39]. The linear response we observed in this study for  0 against water temperature is consistent with studies of developmental rates in the black carp, the winter flounder, and the black plaice [18,20,29,33,35]. However, additional observations are required.
The available data suggest that the graphs of the dependence of  0 on the water temperature are highly species specific. Therefore, the species specificity of the developmental rate can be used to identify the taxonomic ranges of different fish species. We searched the previous study for determining the relationship between  0 and the water salinity. Unfortunately, no previous studies have reported such a relationship between  0 and the water salinity. That's because  0 of several species (having a wide spectrum of salinities) have not been researched so far. So, the future investigation of species having a wide spectrum of salinities needs to focus on the relationship between development and water salinity. The results of this study and an investigation of seeding production techniques based on artificial fertilization, the induction of triploidy to produce sterile organisms, and the induction of tetraploidy, mitotic gynogenetic diploidy, and androgenetic diploidy in the marine medaka will facilitate such research in the future as well.

Conclusion
Considering the identity of the mitotic events and the short time intervals of mitotic intervals ( 0 ), chromosomal manipulation in marine medaka, Oryzias dancena would be the most effective at 0 ppt salinity and water temperatures between 26 and 30°C. This study has demonstrated the specific and clear differences in the rates of hatching, the time of the first cleavage, τ0, and hatching time at different water temperatures and different salinities in the marine medaka. These data will be useful for the development of an optimal treatment protocol for its chromosomal manipulation.
These data on eggs development will make a valuable contribution to a biological aquaculture database.

Ethics Approval and Consent to Participate
The experiments performed in this study complied with the