Rotifer Collection And Culture
The R. rotatoria was sampled at a pond near Jing village (31°73′67′′ N, 118°33′72′′ E) in Hexian County, Anhui Province, China. Then the rotifers were isolated individually under microscope and cultured in the 28℃ constant temperature incubator. The EPA medium, used to maintain rotifers, was freshly prepared by dissolving 96 mg NaHCO3, 60 mg CaSO4, 60 mg MgSO4 and 4 mg KCl in 1 liter of distilled water (pH 7.4–7.8) [58]. The algae Scenedesmus obliquus was grown semi-continuously at 28℃ in an illumination incubator using HB-4 medium [59] and stored at 4 °C for using as rotifer foodstuff after centrifuging in exponential growth. We estimated the density of the algal concentrate by a hemocytometer.
UV-B radiation
The ultraviolet radiation was supplied with UV-B lamps (wavelength range of 280 to 312 nm; UVB-308NM, Shengzheng Guanya Optoelectronic Technology Co., Ltd.). The intensity of UVR was measured by a UV-B radiometer manufactured by Beijing Normal University. In order to ensure that rotifers were under the same UVR intensity, they were placed on the low-speed rotating platform directly below UV-B lamps. Furthermore, to obtain the stable intensity of UV light source, the lamp was turned on 30 minutes before the experiment began. At a certain radiation intensity, the animals will be treated with different doses of UVR by exposing them under UV-B radiation for different time.
Mortality Test
Before starting the experiments, the rotifers were pre-cultured at 28℃ for at least one month to minimize maternal effects. To obtain the value for 50% of the lethal dose (semi-lethal dose, LD50) upon UV-B radiation in three culture volumes of 0.5 ml, 6 ml and 30 ml, we introduced 10 neonates (less than 6 h old) into different culture vessels (0.5-ml culture plates, 6-ml transparent jars and 30-ml beakers respectively) which contained EPA medium with 1.0 × 106 cells/mL of S. obliquus and exposed them to constantly UV-B radiation at the intensity of 0.01, 0.02, 0.03, 0.04 and 0.05 mW/cm2. In total, 540 neonates were tested (10 neonates/treatment × 3 replicates × 3 volumes × 5 UVR intensities, and one control group). The culture vessels were shaded with a quartz cover to allow UV-B transparency and to prevent evaporation during UVB exposure. Thereafter the survival of rotifers was checked every half hour under a stereomicroscope until each individual of every cohort died. Then the LD50 values were respectively derived following the probit method [60].
Life History Experiment
According to the lethal effect assay, to ensure that the rotifer could complete the whole life history, the radiation dose in this experiment were set to 0, 0.012, 0.024, 0.036, 0.048, 0.060, 0.072, 0.084 kJ/m2 at the intensity of 0.01 mW/cm2 (0, 2, 4, 6, 8, 10, 12 and 14 min radiation duration respectively each day). After preculture of one month at 28℃, the neonates (less than 6 h old) were separated indiscriminately and transferred into each well (working volume, 0.5 ml EPA with 1.0 × 106 cells/mL of S. obliquus) of 24-well culture plate. The 24 individuals were inspected for each radiation dose, so 384 neonates in total were prepared and then exposed to UV-B radiation. After UVR treatment, we checked the rotifers every 12 h during the first two days, followed by observation every 6 h until each infant grow into an adult and produced the first offspring. Then they were examined every 12 h until the end of experiment. Meanwhile, the time and number of the offspring produced and the death time of female adult were recorded. During the life table experiment, the surviving mature females were exposed to UV-B radiation at the same time every day, observed and transferred to clean culture plates with fresh food and medium, and the counted neonates were removed.
Population growth of R. rotatoria
At the intensity of 0.02 mW/cm2, the UVR dose was designed to be 0 (control), 0.012, 0.024, 0.048, 0.096, 0.192 and 0.384 kJ/m2 in the population growth experiment (0, 1, 2, 4, 8, 16 and 32 min radiation duration respectively each day). Before the formal experiment, R. rotatoria was pre-cultured at 28℃ for one month. After that, 252 alive neonates (less than 6 h old) were collected and placed equally into 21 glass jars (7 treatments × 3 replicates) containing 6 ml of EPA medium with 1.0 × 106 cells/ml of algae food. These organisms were exposed to UV-B radiation at the same time every day until the end of experiment. To avoid the disadvantageous effects of UVR on algae cells, S. obliquus at the density of 1.0 × 106 cells/ml was fed to rotifers after the radiation treatment once a day. The rotifers were maintained in a 28℃ incubator without light, and counted once every day before the radiation treatment in the first few days, while sampling counted when the population is large.
Determination Of Catalase And Superoxide Dismutase Activity
The radiation dose was designed to be 0 (control), 0.36, 0.72, 1.44 and 2.16 kJ/m2 at the intensity of 0.02 mW/cm2. To meet the demand for detecting the enzyme activity, adequate numbers of rotifer was necessary, and 90 beakers (≈ 3000 individuals in 30-ml beakers) were dealt with in total (6 beakers/treatment × 5 treatments × 3 replicates). After pre-culture as stated above and starvation for one day, rotifers were exposed to the UVR (0, 0.5, 1, 2 and 3 h radiation duration respectively). Then the rotifers were sampling counted and filtered rapidly using a plankton net (mesh size, 20 µm), washed several times with double distilled water, centrifuged, and immediately froze at -80℃ for use within 10 days. The rotifers on ice thawed gradually in a temperature gradient were homogenized by ultrasonication, and centrifuged at 10,000 g for 10 min at 4℃. The supernatant containing the enzyme was collected for the enzymatic assay according to the protocols (SOD by WST-1 method: Cat. No. A001-3; CAT by visible spectrometry method: Cat. No. A007-1; both manufactured by Nanjing Jiancheng Bioengineering Institute, China). The SOD and CAT activities were then measured at the absorbance of 450 nm and 405 nm using an enzyme-labeled meter at 25℃. The soluble protein content in R. rotatoria was measured using Pierce™ BCA Protein Assay Kit (Cat. No. 23227, Thermo Fisher Scientific) according to the protocol direction.
Data analysis
Statistical analysis was performed using SPSS19.0 software. All data were tested for normality using the one-sample Kolmogorov-Smirnov procedure. The homogeneity of variances was checked using Levene’s test. One-way analysis of variance (ANOVA) was conducted to identify the significant effects of UVR on the lethal effects, life history parameters in rotifer, and multiple comparisons were conducted using least significant rank to identify which groups were significantly different among different radiation doses, and the significance level was set to 0.05. Consequently, regression analysis was performed on the relationships of the dose–response.
The durations of juvenile period (JP, the time between a neonate and that producing the first baby), reproductive period (RP, the time between an adult producing the first baby and the last baby), longevity and offspring of rotifers grown at different doses of UV-B radiation were calculated depended on the recorded data. In the population growth experiment, the population growth rate was calculated using the formula , where N0 is the initial population density (2 ind./ml), and Nt is the population density at time t in days [61].