2 Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
3 University of Chinese Academy of Sciences, Beijing 100049, China;
4 Jiaozhou Bay Marine Ecosystem Research Station, Chinese Academy of Sciences, Qingdao 266071, China;
5 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China;
6 Department of Engineering and Technology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
Marine zooplankton live in a continuously changing environment. Light is one of the most influential environmental factors for marine zooplankton. The change of light consists of two aspects: intensity and spectrum. Many planktonic species exhibit significant diel vertical migration in the seawater column, a phenomenon mainly regulated by light (Ewald, 1910; Ringelberg, 1964, 1999; Forward, 1988; Tao et al., 2004). Diurnal solar irradiation is a fundamental ecological factor in the marine ecosystem, playing a key role in controlling thermal stratification and water-column mixing, thereby affecting the entire marine food web. Thus, at the global scale, many marine processes are influenced by sunlight, such as the marine carbon and biogenic dimethylsulfide cycles (Sunda and Huntsman, 1997; Farquhar and Roderick, 2003; Vallina and Simo, 2007). The spectral composition of sunlight at the earth's surface consists mainly of visible light and ultraviolet A, B, and C (UV-A, UV-B and UV-C). Among the ultraviolet wavelengths, UV-B has the greatest impact on marine zooplankton. Jerlov (1950) confirmed the potential hazard of ultraviolet radiation to marine organisms after measuring UV-B irradiation to depths of 20 m in the eastern Mediterranean Sea. In the past decades, several studies have shown that a decrease in stratospheric ozone allows an increased flux of UV-B radiation to reach the earth's surface (Madronich et al., 1995). Moreover, numerous studies have reported that environmental pollution causes serious damage to the ozone layer, as a considerable amount of UV-B radiation increasingly reaches the Earth's surface, especially at middle latitudes (Smith et al., 1992; Kerr and McElroy, 1993). Increases in UV radiation can directly affect the behavior of zooplankton, thereby indirectly affecting the marine food web (Cullen and Neale, 1994; Karanas et al., 1979; Araseth and Schram, 1999; Mostajir et al., 1999; Speekmann et al., 2000; Piazena et al., 2002).
The planktonic copepod C. sinicus Brodsky 1962 is widely distributed in margins of the western North Pacific from Japan to Vietnam (Chen, 1964; Anon, 1977; Kidachi, 1979a, b ; Huang et al., 1993). It is the dominant and key zooplankton species in the Yellow Sea and the East China Sea, and it was chosen as the target species in the China-Global Ocean Ecosystem Dynamics research program (Sun et al., 2002). Moreover, C. sinicus is generally regarded as one of the most important zooplankton species in shelf waters by virtue of its enormous abundance, large body size, and significant role in the conversion of primary production to higher trophic levels (Lin and Li, 1986; Uye et al., 1986). The mature female C. sinicus can be found throughout the year in the Yellow Sea, but its population reproduction and recruitment are mainly in spring. Its distribution pattern, life cycle and population dynamics are in relation to many physical environments (Wang et al., 2003, 2009; Zhang et al., 2005). Significant activities of this species, such as its diel vertical migration (DVM) and tendencies to graze at night and lay eggs near dawn, are presumably related with the diel light cycle.
We conducted laboratory experiments to study the behavioral responses of C. sinicus exposed to artificially generated, wavelength-specific visible light and ultraviolet light (UV-B, 280–315 nm). The aim of the study was to characterize the photobehavior of C. sinicus, specifically phototaxis, UV-B-induced mortality, and the influence of light on its grazing and reproduction activities.2 MATERIAL AND METHOD 2.1 Sampling and culture
The zooplankton used in the experiments was collected in the Yellow Sea, using a conical macrozooplankton net (mesh size 500 μm, diameter 80 cm), towed vertically, from 2 m above the bottom and up to the surface, at a rate of about 0.8 m/s. The mature female C. sinicus for the egg production experiment were collected during May 2004 in Jiaozhou Bay in the Yellow Sea (120.25°E 36.10°N, depth: 18 m). Other experiments were conducted from December 2003 to April 2004 in the Yellow Sea (123°E 35°N, depth: 70 m). The sampling contents of the cod end were transported to the laboratory in 10-L plastic buckets filled with pretreated seawater (10–15 ℃, which was the in situ seawater temperature). Actively swimming and visually healthy adult C. sinicus were identified and picked out under a stereo zoom microscope (Nikon SMZ-745T, Japan). Before the phototaxis and other incubation experiments, the selected C. sinicus were kept in the dark for less than 1 h with an aim to minimize stress. Meanwhile, the preliminary tasks of the experiments will be completed during this period.2.2 Photosensitivity of Calanus sinicus
Visible light was generated using a metal halide fiber illuminator (Mejiro Precision BMH-250, Japan), which can produce spots of uniform light intensity by transferring light to a compound lens via the optical fiber. The intensity of ultraviolet in the light spectrum produced by this device is very low, and the waveband of visible light in the spectrum is close to that of natural sunlight. The combination of a narrow-band filter and compound lens gives rise to the monochromatic light of different wavebands; hence, with the half-width of the filter being less than 10 nm, seven wave bands were selected (Table 1).
Thirty adult C. sinicus were placed into partitioned area C of the experimental device shown in Fig. 1. The individuals were allowed 5 min to adapt to dark conditions, to eliminate external interference on their phototaxis (Fig. 1a). Next, the horizontal partition plate was removed and the C. sinicus were irradiated (from the left side of the device) with either visible light or ultraviolet rays of various wavelengths and intensities (Fig. 1b). After irradiation for 3 min, the partition plate was reinstalled and the light source halted. The numbers of C. sinicus in each partition area were then counted (Fig. 1c). As a control, the entire procedure was repeated using another 30 individuals of C. sinicus and all dark conditions in the same device. Each individual C. sinicus was used only once in the experiment.
Six different intensities were projected for each waveband (Table 1). It was expected that the phototaxis would cause the C. sinicus to move toward the partitioned area nearest the light source or to the partitioned areas separated from the light source by two partitions. Photosensitivity was determined by measuring the phototactic response of the copepods. Phototaxis is defined as directional in relation to a directional light source: a positive response is toward the light source, while movement away is negative (Stearns and Forward, 1984). Therefore, the phototaxis of C. sinicus in the partitioned area nearest the light source was deemed positive phototaxis, and that in the partition farthest from the light source was deemed negative phototaxis. Phototaxis ratio (PR) was expressed as the ratio (%) of the number of C. sinicus in the partitioned area nearest or farthest from the light source versus the total number. Phototaxis index (PI) was expressed as the positive phototaxis ratio (PRp) minus the negative phototaxis ratio (PRn), namely PI=PRp−PRn. The phototaxis was divided into five grades based on the determination of the PI, as follows: -1.0 to -0.6, strong negative phototaxis; -0.6 to -0.2, weak negative phototaxis; -0.2 to 0.2, no phototaxis; 0.2 to 0.6, weak positive phototaxis; and 0.6–1.0, strong positive phototaxis. The waveband with the least light intensity was considered to be the one to which the C. sinicus were the most sensitive under conditions of the same PI.
We conducted exposure experiments using laboratory-generated ultraviolet light (UV-B, 280– 315 nm) to evaluate the phototaxis and tolerance of C. sinicus to UV-B. The lethal time of different UV-B intensities on C. sinicus was determined using a UV fiber optical transmission system (Mejiro Precision CHG-200, Japan).2.3 Light-induced grazing behavior
For this experiment, zooplankton in the laboratory was reared on a mixed algal diet consisting of three kinds of algae (Skeletonema costatum, Platymonas subordiformis, and Phaeodactylum tricornutum) at the ratio 1:1:1 in terms of carbon concentrations. The algal mix, which grew well after being cultured for 4–6 days, was fed to the C. sinicus at a final diet concentration of 0.42–1.25 μg C/mL, which has been previously determined to satisfy the needs of C. sinicus (Frost, 1972; Uye, 1986).
Thirty adult C. sinicus were irradiated with different intensities of wavelength-specific visible light and UV-B, for periods of 20 min. After switching off the given light source, the fecal pellets of the copepods were then counted under a Nikon microscope. As controls, the same procedure was repeated using another 30 C. sinicus in dark conditions. The intensities of the red (649 nm), orange (594.9 nm), yellow (576.8 nm), green (545.8 nm), cyan (509.2 nm), blue (488.6 nm) and purple (415.3 nm) light were 0.121, 0.668, 1.475, 1.375, 0.116, 0.223, and 0.038 mW/cm2, respectively. These experiments were done in triplicate for each intensity of light wavelength, and the results of each experiment were expressed as the mean of the three measurements. The number of fecal pellets produced by copepods under the different light intensities reflected the influence of light in different wavebands on the ingestion of C. sinicus. Fecal pellet production rate (FPPR) indicated the mean amount of feces discharged of one individual C. sinicus within a unit of time.2.4 Effects of light on spawning and hatching
The zooplankton spawning device used in this experiment is a patented device developed by our laboratory; the device can effectively prevent adult zooplankton from consuming the eggs they spawn (Fig. 2). Sexually mature female C. sinicus were chosen and then cultured in dark conditions for 48 h so that they could adapt to the environment. Five female C. sinicus were placed into each spawning bottle; an experimental group contained five bottles; in advance, 200 mL of filtered seawater was prepared to fill each bottle. The bottles were irradiated by different intensity white light for a period of nine continuous days in thermostat incubators. Every 24 h, eggs of the C. sinicus were removed from each bottle along with a portion of the seawater, via a special switch in the bottom of the device, and then the bottles were supplemented with seawater of the same volume, temperature, and concentration of algal diet. The diet mixture of three kinds of algae aimed to eliminate the influence of the algal type on the copepods' egg production and hatching rates (Li et al., 2006). The dietary algae concentration was 1.02±0.1 μg C/mL. The entire duration of the spawning experiment and hatching experiment were performed in thermostat incubators, and the experimental water temperature was the same as that of the seawater from which individual C. sinicus was originally collected, and thus maintained at 13.6±0.5 ℃.
Eggs in each experimental group were counted under the StereoZoom microscope to calculate the daily egg production rate (EPR) of C. sinicus exposed to different light intensities. The EPR indicates the number of eggs produced by a female C. sinicus in one day, presented as the mean value from five parallel experimental groups. Differences in the EPRs reflect the effects of light of different intensities on the spawning behavior of C. sinicus (i.e., the EPR is in direct proportion to the suitability of the corresponding light intensity for the spawning of C. sinicus).
In the egg production experiment, eggs produced by all the female C. sinicus within 24 h were pooled, and then 30 eggs were taken out at random (all eggs were taken in cases where the total number of eggs was less than 30). The eggs were put in a 100-mL beaker containing 80 mL of filtered seawater, to allow hatching of the eggs in the breaker for 48 h under dark and thermostatic conditions (the temperature being the same with that for spawning), respectively. Nauplii and non-hatched eggs were counted under a StereoZoom microscope to calculate the hatching rate, indicated by the ratio (%) of nauplii to all eggs available for potential hatching. Differences in the hatching rates reflect the effect of different light intensities on the hatching behavior of C. sinicus. Zhang et al. (2002a) considered that 43.5 h was needed for C. sinicus to achieve a maximal hatching rate at 16 ℃. The hatching duration for C. sinicus was determined to be 12 h at 20 ℃, and 24 h at 14 ℃ (Zhang et al., 2002b). In the current experiment, the temperature was 13.6±0.5 ℃, and therefore 48 h was deemed adequate for hatching out spawns of C.sinicus.3 RESULT 3.1 Phototaxis of Calanus sinicus under different light spectra
During the phototaxis experiment, the multiple control experiments that were performed in dark conditions demonstrated that for C. sinicus cultured indoors, the positive and negative phototaxis ratio could be kept at about 40%, hence the positive phototaxis ratio did not differ significantly from the negative phototaxis ratio.
Phototaxis of C. sinicus captured in the field and subjected to visible light in the laboratory: The PI of C. sinicus was -0.70−-0.37 in the case of white light throughout the spectrum at intensities of 4.72–10.50 mW/cm2, manifesting a negative phototaxis. Calanus sinicus showed weak positive phototaxis (PI=0.27) to 1.09 mW/cm2 white light. The PI was within the range 0.23 to 0.87 in the case of 488.6 nm blue light at intensities of 0.018–0.155 mW/cm2, 509.2 nm green light at intensities of 0.009–0.080 mW/cm2 and 576.8 nm yellow light at intensities of 0.533–1.155 mW/cm2, manifesting a positive phototaxis. In the case of 594.9 nm orange light, the highest PI were -0.13 at the lowest intensity (0.058 mW/cm2) and the other PI was lower than -0.40 at intensities of 0.136–0.489 mW/cm2, thus manifesting a negative phototaxis. In the case of 415.3 nm purple light, 545.8 nm green light, and 649.0 nm red light, the PI was mostly between -0.2 and 0.2, and accordingly manifested no phototaxis. According to the average PI of different intensities white light and seven monochromatic light, C. sinicus showed positive phototaxis to blue (average PI=0.67), cyan (average PI=0.46) and yellow (average PI=0.36) light, and negative phototaxis to orange (average PI=-0.43) and white (average PI=-0.31) light (Fig. 3).
Pre-tests demonstrated that the lowest intensity of UV-B that may be sensed by C. sinicus is under 0.016 mW/cm2; therefore, the UV-B intensity was tested at 0.005, 0.010, and 0.016 mW/cm2, and three parallel experiments were performed for each waveband and light intensity. Calanus sinicus manifested weak negative phototaxis in response to UV-B intensities set at 0.010 mW/cm2 and 0.016 mW/cm2, with a resultant PI of -0.38 and -0.49, respectively. Calanus sinicus showed no apparent phototaxis (PI=-0.18) to the UV-B intensity of 0.005 mW/cm2. Together, these results revealed that C. sinicus is able to sense the presence of UV-B radiation and avoid its harmful effects through negative phototaxis.3.2 The lethal effect of UV-B on Calanus sinicus
Results of the experiment on the potentially lethal effect of UV-B on C. sinicus demonstrated that this species possesses a good defensive response against UV-B intensities of less than 0.10 mW/cm2, whereas UV-B intensities higher than those in nature (generally, < 0.30 mW/cm2) showed a lethal action on the copepods. The UV-B lethal time (LT50) indicates the time to 50% mortality as a result of the particular UV-B intensity. A lineal regression was adopted to calculate the LT50 of C. sinicus exposed to UV-B of different intensities (Fig. 4). For the UV-B intensity of 0.020 mW/cm2, the equation was y=0.017x−0.018, R2=0.567, resulting in LT50=30.47 h. The LT50 of C. sinicus was 2.86 and 1.96 h under UV-B intensity of 0.030 and 0.050 mW/cm2.3.3 Grazing rates of Calanus sinicus under visible light and UV-B
For C. sinicus irradiated by white light throughout the spectrum with different intensities and for the controls in all dark conditions, the calculated FPPRs were stable, at approximately 6 fecal pellets/(ind.·h), and thus did not significantly differ. Therefore, short durations (20 min) of white-light irradiation throughout the spectrum did not significantly affect the copepods' ingestion.
In the experiments using 415.3, 488.6, 509.2, and 545.8 nm monochromatic light, neither the experimental groups under the different light conditions or the control group under the all dark conditions manifested a high FPPR. The mean FPPR was 12 fecal pellets/(ind.·h) in the case of all monochromatic light types and all intensities, a result attributed to the relatively short culture time. In the experiments using the other five monochromatic light types, the FPPRs of the C. sinicus dropped more or less due to the action of the culture time, with mean FPPRs of 5–11 fecal pellets/(ind.·h). In the case of irradiation with different wavelengths and intensities, the FPPRs of the experimental groups were likewise comparable with that of the controls. The experimental group differed significantly from the control group only in the case of yellow, orange and red light, and the FPPR was relatively stimulated; furthermore, the intensity of yellow light was in direct proportion to the estimated FPPR (Fig. 5). These results show that C. sinicus ingested slightly but statistically significantly more food under the action the higher intensities of yellow-red light, whereas their ingestion was not significantly affected under the other monochromatic light types.
Irradiation with UV-B at intensities of 0.10, 0.20, and 0.40 mW/cm2 significantly inhibited the copepods' ingestion: the highest FPPR in a treatment group was a mere 3.6 fecal pellets/(ind.·h), while the lowest FPPR of a control group was 4.8 fecal pellets/(ind.·h), and the difference was very significant (P < 0.01) (Table 2). We suggest that UV-B at the three different intensities did not significantly affect the ingestion of C. sinicus.3.4 Egg production and hatching rates of Calanus sinicus under different white-light intensities
In the 9-day spawning experiment, the highest EPRs (the mean value from five parallel experiments) occurred on Day 5, both for the control group in dark conditions and for an experimental group in light conditions. The highest EPR for C. sinicus in all dark conditions was 9.89 eggs/(female·d), while the mean EPR in the whole spawning experiment was 4.12 eggs/(female·d). The highest EPR was 20.28 eggs/(female·d) under a white-light intensity of 1.58 mW/cm2, while the mean EPR nine days later was 10.04 eggs/(female·d). During the nine days, C. sinicus showed the highest single EPR and the highest mean EPR under an intensity of 1.58 mW/cm2 (Fig. 6). The spawning rate of C. sinicus was always higher under light conditions than under dark conditions, demonstrating that irradiation may promote spawning of C. sinicus, and indeed that illumination may be essential for C. sinicus to spawning.
Spawning of the C. sinicus under the different irradiation conditions showed that the hatching rate did not differ significantly among different light conditions within the first three days; furthermore, the hatching rate was relatively high under all conditions, always exceeding 75%. The hatching rates under both light and dark conditions began to change significantly on Day 4; the hatching rate was higher for spawns produced either in dark conditions or under lower light intensities (0.78 and 1.58 mW/cm2) than that produced under high light intensity (2.53 mW/cm2) (Fig. 7).4 DISCUSSION 4.1 Phototaxis and diel vertical migration of Calanus sinicus
Ewald (1910) was the first to record that planktonic zooplankton mainly inhabits deeper water during the day and ascend to upper water layers at night. Accordingly, he hypothesized that light affects the diel vertical migration (DVM of the zooplankton. There are two general hypotheses about how light might regulate the DVM of planktonic zooplankton. The preferendum hypothesis states that this migration of zooplankton occurs as they search for the optimum light intensity (Ewald, 1910; Russell, 1926, 1934; Boden and Kampa, 1967). The relative-stimulus threshold hypothesis proposes that the change of light in the day's changing environment is a signal for zooplankton to determine whether vertical migration should occur (Clarke, 1933; Ringelberg, 1964; Daan and Ringelberg, 1969; Buchanan and Haney, 1980; Haney et al., 1990). Ultraviolet radiation is harmful to zooplankton (Klugh, 1929; Dey et al., 1988; Williamson et al., 1994; Chalker-Scott, 1995; Zagarese et al., 1997; Storz and Paul, 1998), thus many researchers have suggested that the tendency of zooplankton to stay in deeper water during the day is a mechanism limiting their exposure to the ultraviolet radiation of sunlight (Williamson et al., 1994; Araseth and Schram, 1999; Speekmann et al., 2000; Leech and Williamson, 2001; Rhode et al., 2001).
As the dominant and key zooplankton species in the Yellow Sea and the East China Sea, C. sinicus shows obvious DVM behavior (Uye et al., 1992; Wang et al., 2003; Zuo et al., 2004). Jékely et al. (2008) found that the tiny marine annelid Platynereis dumerilii could mediate phototactic swimming using their simple eyespots, with the mechanism of its phototaxis based on molecular tools; this indicated that zooplankton could feel and distinguish light and consequently adjust their behaviors. Stearns and Forward (1984) studied the phototaxis of another marine copepod, Acartia tonsa, and found that mature females manifest positive phototaxis in response to light, with a greater degree of phototaxis with increasing light intensities; A. tonsa was relatively sensitive to visible light at 453–620 nm, with phototaxis strongest in response to 580 nm visible light. The sensitivity of A. tonsa to visible light at wavelengths outside the range of 453–620 nm dropped remarkably; however, A. tonsa was still highly sensitive to 380 nm and 700 nm visible light as long as the intensity was strong enough. Sweatt and Forward (1985) found that the planktonic chaetognath Sagitta hispida was most sensitive to green-blue light, especially at wavelengths of 500 nm. Smith and Macagno (1990) found that the relatively large-sized freshwater water flea Daphnia magna was fairly sensitive to 434 nm, 525 nm, and 608 nm visible light, and to 348 nm ultraviolet. In our experiments, C. sinicus developed noticeable positive phototaxis in response to 488.3 nm and 509.2 nm blue-cyan light, a result which agrees with Forward's (1988) conclusion that zooplankton is most sensitive to blue-cyan light. Wang et al. (2006) found that the absorption coefficients of 488–555 nm light spectra were highest in the Yellow Sea. The C. sinicus were relatively sensitive to blue-cyan and yellow light, manifesting relatively strong positive phototaxis, possibly because, as a monochromic light, blue-cyan light penetrates farthest into the marine waters of its natural region, and thus blue-cyan light may act as a chief signal for the DVM of C. sinicus.4.2 Effects of UV-B on photoresponse of Calanus sinicus
In the eastern Mediterranean Sea, at a middle latitude, the intensity of 310 nm ultraviolet decreased by 14% as depth increased by 1 m, and the intensity of 375 nm ultraviolet decreased by 5% (Jerlov, 1950). If the intensity of UV-B was 0.20 mW/cm2at sea level at noon, it would be approximately 0.01 mW/cm2 at a depth of 20 m; the latter UV-B intensity may be the degree at which it is sensed by C. sinicus. In addition, the UV-B intensity in the morning just as the sun rises is also approximately 10 mW/cm2. Therefore, it may be concluded that ultraviolet radiation is an important cause and signal for the DVM of C. sinicus. For instance, Rhode et al. (2001) showed that several species of Daphnia would descend to deeper water when being irradiated by ultraviolet, again implicating ultraviolet as a primary factor responsible for the DVM of zooplankton. In addition, those authors considered that the DVM was a behavior formed under the synergic action of multiple factors. Schulyer and Sullivan (1997) also suggested that zooplankton can sense ultraviolet. Storz and Paul (1998) found that species of Daphnia manifest a negative phototaxis in response to ultraviolet irradiation. Araseth and Schram (1999) demonstrated that the North Atlantic copepod C. fimarchicus could sense ultraviolet and also distinguish ultraviolet from visible light, manifesting negative phototaxis in response to ultraviolet. Speekmann et al. (2000) studied the behavior of Acartiura spp., Acanthacartia spp., and Tortanus dextrilobatus under the action of ultraviolet, and reported that early larvae of T. dextrilobatus and C. pallasi were sensitive to ultraviolet and manifested noticeable harm-avoiding behavior by changing their vertical distribution (i.e., they would remain at 50 cm deeper than the normal depth free of ultraviolet); in addition, the mortality of T. dextrilobatus increased remarkably when the copepods were fully exposed to ultraviolet. Leech and Williamson (2001) demonstrated that as the surface water was subjected to strong ultraviolet radiation at noon and in the afternoon, most individuals of Daphnia would move to lower water layers, but then they would return to the upper layer as soon as the ultraviolet was shielded. Klugh (1929) pointed out that ultraviolet radiation could be lethal to copepods. That conclusion was eventually verified by the indoor experiments of Dey et al. (1988) and in freshwater environments (Williamson et al., 1994). Zagarese et al. (1997) studied the action of ultraviolet on three species of Boeckella (B. brevicaudata, B. gibbosa and B. gracilipes), and found that not all of these zooplankton species were necessarily injured or killed by ultraviolet, but only B. gracilipes was exceptionally sensitive and fragile to ultraviolet light conditions, while the other two Boeckella species possessed a good ultraviolet-defense mechanism.
The current study demonstrated a pronounced lethal action of UV-B on C. sinicus when it was artificially generated at higher intensities than found in the natural environment, but that C. sinicus appear to possess a good preventive mechanism against UV-B at intensities lower than the natural intensities. However, the intensity of UV-B that might be sensed by C. sinicus (~0.010 mW/cm2) was far lower than the intensity that may cause injury to C. sinicus (~0.200 mW/cm2), showing that the copepods sensed the presence of UV-B and then escaped it before it could cause injury.4.3 Grazing behavior of Calanus sinicus under different light conditions
According to both field and laboratory experiments, diel grazing is found in many zooplankton species (Zheng and Zheng, 1989; Wang et al., 1998; Strom, 2001). A great number of studies have used feces as an index of the ingestion of zooplankton. Though the FPPR could not be used to calculate the absolute amount of food ingested by the copepods, it might reasonably reflect how ingestion changes along with the condition of light irradiation. Because feces are formed from the ingested feedstuff, the method based on the FPPR should be reliable (Zhang and Wang, 2000).
Zhang and Wang (2000) considered that an individual of C. sinicus might discharge 5–10 fecal pellets in an hour when sufficient food is available, an approximation which agrees with the results of the current experiment. In contrast, Karanas et al. (1979) reported that the ingestion rate of common copepods (such as Calanoida) was higher under dark conditions, and dropped with increasing light intensity in indoor light conditions, which disagrees with the results of the current experiment. One explanation may be as follows: first, the C. sinicus did not immediately respond to the irradiation conditions while they ingested food, but rather delayed their response, granting that the duration of the current experiment was relatively short; second, the current experiment adopted relatively high intensities of the white light throughout the spectrum. Zheng and Zheng (1989) made continuous observations of the diurnal ingestion behavior of Calanopia thompsoni and found a low rate of ingestion during the daytime, as proven by almost empty stomachs, while ingestion tended to increase gradually in the evening and in the small hours of the night. Such ingestion behavior has been similarly observed in other copepods (for example, in Centropages). Wang et al. (1998) examined the intestinal contents of copepods and found that as a rule, individual copepods exhibited diurnal ingestion, which was manifested especially in larger-sized zooplankton. The peak of intestinal content occurred from dusk to midnight (18:00–24:00), and the peak intestinal content could be 10 times the minimal intestinal content found in the daytime. This may be because most zooplankton is characterized by DVM and ingest food after dusk and in the early hours of the night.
The experiment on the influence of UV-B on the ingestion of C. sinicus showed that high-intensity UV-B manifested a strong inhibiting action on C. sinicus. In the present work, we have demonstrated that C. sinicus shows negative phototaxis to UV-B even at the low intensity of 0.010 mW/cm2; accordingly, C. sinicus appears to possess a good preventive mechanism against UV-B, as demonstrated when continuous irradiation with 0.100 mW/cm2 UV-B for 4 h did not cause the death of any individual copepod. Based on this, it may be deduced that within the range of 0.010–0.100 mW/cm2 there is likely a critical intensity that ultimately affects the ingestion rate of C.sinicus; this possibility offers a good theoretical basis for further studies of the DVM of C. sinicus.4.4 Effects of visible light on the reproduction of Calanus sinicus
The spawning and hatching rates of zooplankton are important parameters in marine ecology studies, and numerous investigations of this topic have been made in the past 20 years. Most recent research efforts have emphasized how light indirectly affects the spawning and hatching of zooplankton via direct action on the phytoplankton biomass, as well as the relationship between the light cycle and the formation of diapause spawns and dormant spawns; however, no study to date has investigated the direct effects of light.
Lin and Li (1986) studied the daily egg production of C. sinicus and noticed that the species' spawning mode followed a pattern: the C. sinicus spawned for six consecutive days, then suspended spawning for four or five days, and then resumed spawning. Zhang et al. (2002a) considered that C. sinicus may successively spawn for 35 days under a state of satiation; in addition, C. sinicus at peak spawning may spawn for nine consecutive days before an intermission lasting at most two days. The disagreement in the findings of these two studies may be attributable to the different sites where the C.sinicus individuals were originally collected. More extensive studies could clarify whether the oogonia of C. sinicus grow steadily, and what conditions might affect their growth. The results of the current study are in basic agreement with several previous studies, although differences exist due to differences in regions and temperature. Schulze (1928) conducted the first study of the spawning and hatching of zooplankton under different light conditions and found that the reproductive rate of Daphnia pulex dropped under dark conditions, which agrees with our results, but is contrary to the observation that most zooplankton species spawn at night (Uye et al., 1990; Atkinson et al., 1996; Wang et al., 1998). Through a field experiment in the Yellow Sea, Zhang et al. (2002b) found that the spawning of C. sinicus showed a noticeable diurnal rhythm: the great majority of C. sinicus individuals moved to the upper layers of seawater to spawn from approximately midnight to 03:00–06:00, and most spawns occurred near 06:00, all findings which agree with the results of the current experiments.
Spawns of C. sinicus produced under different light conditions did not differ significantly regarding hatching rate in the early stage of the experiment (the first 3 days), which may constitute a delayed action of the irradiation on the species' spawning. The hatching rate of spawns of C. sinicus produced under white light at a high intensity was lower than that of spawns produced under dark conditions or low-light conditions, indicating that high-intensity white light may lower the spawning quality.5 CONCLUSION
Calanus sinicus distributed in the Yellow Sea showed positive phototaxis to the blue and cyan light but negative phototaxis to orange light and UV-B (≥0.01 mW/cm2). High UV-B radiation (≥0.20 mW/cm2 UV-B) causes a significant lethal effect on the copepod. The yellow-red light stimulates the grazing of C. sinicus. The egg production rate of C. sinicus was highest under a white-light intensity of 1.58 mW/cm2, which are consistent with the observed phenomenon that C. sinicus in the Yellow Sea mostly spawn near dawn. Both the light intensity and spectrum are the most important factors influencing the diel vertical migration of C. sinicus in the Yellow Sea.6 DATA AVAILABILITY STATEMENT
The data used in the current study are available from the corresponding author on reasonable request.7 ACKNOWLEDGMENT
We thank the crew of the R/V Beidou for the assistance in zooplankton sampling during the field survey.
Anon. 1977. Study on plankton in China Seas. In: Anon ed.Scientific Reports of "Comprehensive Oceanography Expedition in China Seas", Ocean Res. Off. Press, Tianjun, 8: 1-159. (in Chinese)
Araseth K A, Schram T A. 1999. Wavelength-specific behaviour in Lepeophtheirus salmonis and Calanus finmarchicus to ultraviolet and visible light in laboratory experiments (Crustacea:Copepoda). Marine Ecology Progress Series, 186: 211-217. DOI:10.3354/meps186211
Atkinson A, Ward P, Murphy E J. 1996. Diel periodicity of subantarctic copepods:relationships between vertical migration, gut fullness and gut evacuation rate. Journal of Plankton Research, 18(8): 1 387-1 405. DOI:10.1093/plankt/18.8.1387
Boden B P, Kampa E M. 1967. The influence of natural light on the vertical migrations of an animal community in the sea. Symposia of the Zoological Society of London, 19: 15-26.
Buchanan C, Haney J F. 1980. Vertical migration of zooplankton in the Arctic. A test of the environmental controls. In:Kerfoot W C ed. Evolution and ecology of Zooplankton communities. New Hampshire:University Press of New England, 3:66-79. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_2657746
Chen Q C. 1964. A study of ratio and the breeding periods, variation in sex in size of Calanus sinicus brodsky. Oceanologia et Limnologia Sinica, 6(3): 272-287. (in Chinese with English abstract)
Clarke G L. 1933. Diurnal migration of plankton in the Gulf of Maine and its correlation with changes in submarine irradiation. Biological Bulletin, 65(3): 402-436. DOI:10.2307/1537215
Cullen J J, Neale P J. 1994. Ultraviolet radiation, ozone depletion, and marine photosynthesis. Photosynthesis Research, 39(3): 303-320. DOI:10.1007/BF00014589
Daan N, Ringelberg J. 1969. Further studies on the positive and negative phototactic reaction of Daphnia magna Straus. Netherlands Journal of Zoology, 19(4): 525-540.
Dey D B, Damkaer D M, Heron G A. 1988. UV-B dose/doserate responses of seasonally abundant copepods of Puget Sound. Oecologia, 76(3): 321-329. DOI:10.1007/BF00377024
Ewald W F. 1910. Über Orientierung, Lokomotion und Lichtreaktionen einiger Cladoceren und deren Bedeutung für die Theorie der Tropismen. Biologisches Zentralblatt, 30: 1-16.
Farquhar G D, Roderick M L. 2003. Atmospheric science:Pinatubo, diffuse light, and the carbon cycle. Science, 299(5615): 1 997-1 998. DOI:10.1126/science.1080681
Forward R B Jr. 1988. Diel vertical migration:zooplankton photobiology and behaviour. Oceanography and Marine Biology, An Annual Review, 26: 361-393.
Frost B W. 1972. Effects of size and concentration of food particles on the feeding behavior of the marine planktonic copepod Calanus pacificus. Limnology and Oceanography, 17(6): 805-815. DOI:10.4319/lo.1972.17.6.0805
Haney J F, Craggy A, Kimball K, Weeks F. 1990. Light control of evening vertical migrations by Chaoborus punctipennis larvae. Limnology and Oceanography, 35(5): 1 068-1 078. DOI:10.4319/lo.1918.104.22.1688
Huang C, Uye S, Onbé T. 1993. Geographic distribution, seasonal life cycle, biomass and production of a planktonic copepod Calarms sinicus in the inland Sea of Japan and its neighboring Pacific Ocean. Journal of Plankton Research, 15(11): 1 229-1 246. DOI:10.1093/plankt/15.11.1229
Jékely G, Colombelli J, Hausen H, Guy K, Stelzer E, Nédélec F, Arendt D. 2008. Mechanism of phototaxis in marine zooplankton. Nature, 456(7220): 395-399. DOI:10.1038/nature07590
Jerlov N G. 1950. Ultra-violet radiation in the sea. Nature, 166(4211): 111-112. DOI:10.1038/166111a0
Karanas J J, Van Dyke H, Worrest R C. 1979. Midultraviolet(UV-B) sensitivity of Acartia clausii Giesbrecht(Copepoda). Limnology and Oceanography, 24(6): 1 104-1 116. DOI:10.4319/lo.1922.214.171.1244
Kerr J B, McElroy C T. 1993. Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion. Science, 262(5136): 1 032-1 034. DOI:10.1126/science.262.5136.1032
Kidachi T. 1979a. Systematics of Calanus in the Japanese waters, with special reference to morphological differentiations of Calanus in Sagami Bay, part 1. Aquabiology, 2: 9-15.
Kidachi T. 1979b. Systematics of Calanus in the Japanese waters, with special reference to morphological differentiations of Calanus in Sagami Bay, part 2. Aquabiology, 3: 25-31.
Klugh A B. 1929. The effect of the ultra-violet component of sunlight on certain marine organisms. Canadian Journal of Research, 1(1): 100-109. DOI:10.1139/cjr29-006
Leech D M, Williamson C E. 2001. In situ exposure to ultraviolet radiation alters the depth distribution of Daphnia. Limnology and Oceanography, 46(2): 416-420. DOI:10.4319/lo.2001.46.2.0416
Li J, Sun S, Li C L, Zhang Z, Tao Z C. 2006. Effects of single and mixed diatom diets on the reproduction of copepod Calanus sinicus. Acta Hydrochimica et Hydrobiologica, 34(1-2): 117-125. DOI:10.1002/(ISSN)1521-401X
Lin Y S, Li S. 1986. Laboratory survey on egg production of marine planktonic copepod Calanus sinicus in Xiamen Harbour. Journal of Xiamen University (Natural science), 25(1): 107-112. (in Chinese with English abstract)
Madronich S, Mckenzie R L, Caldwell M, Bjorn L O. 1995.Changes in ultraviolet-radiation reaching the Earth's surface. Environmental Effects of Stratospheric Ozone Depletion, 1994 Update.
Mostajir B, Demers S, De Mora S D, Belzile C, Chanut J P, Gosselin M, Roy S, Villegas P Z, Fauchot J, Bouchard J, Bird D, Monfort P, Levasseur M. 1999. Experimental test of the effect of ultraviolet-B radiation in a planktonic community. Limnology and Oceanography, 44(3): 586-596. DOI:10.4319/lo.1999.44.3.0586
Piazena H, Perez-Rodrigues E, Häder D P, Lopez-Figueroa F. 2002. Penetration of solar radiation into the water column of the central subtropical Atlantic ocean-optical properties and possible biological consequences. Deep Sea Research Part Ⅱ:Topical Studies in Oceanography, 49(17): 3 513-3 528. DOI:10.1016/S0967-0645(02)00093-0
Rhode S C, Pawlowski M, Tollrian R. 2001. The impact of ultraviolet radiation on the vertical distribution of zooplankton of the genus Daphnia. Nature, 412(6842): 69-72. DOI:10.1038/35083567
Ringelberg J. 1964. The positively phototactic reaction of Daphnia magna Straus:a contribution to the understanding of diurnal vertical migration. Netherlands Journal of Sea Research, 2(3): 319-406. DOI:10.1016/0077-7579(64)90001-8
Ringelberg J. 1999. The photobehaviour of Daphnia spp. as a model to explain diel vertical migration in zooplankton. Biological Reviews of the Cambridge Philosophical Society, 74(4): 397-423. DOI:10.1017/S0006323199005381
Russell F S. 1926. The vertical distribution of marine macroplankton. Ⅳ. The apparent importance of light intensity as a controlling factor in the behaviour of certain species in the Plymouth area. Journal of the Marine Biological Association of the UK, 14: 415-440. DOI:10.1017/S0025315400007918
Russell F S. 1934. The vertical distribution of marine macroplankton. 12. Some observations on the vertical distribution of Calanus finmarchicus in relation to light intensity. Journal of the Marine Biological Association of the UK, 19: 569-584. DOI:10.1017/S0025315400046646
Schulyer Q, Sullivan K B. 1997. Light responses and diel migration of scyphomedusa Chrysaora quinquecirrha in mesocosms. Journal of Plankton Research, 19(10): 1 417-1 428. DOI:10.1093/plankt/19.10.1417
Schulz H. 1928. Über die Bedeutung des Lichtes im Leben niederer Krebse. Zeitschrift für Vergleichende Physiologie, 7(3): 488-552. DOI:10.1007/BF00339028
Scott L C. 1995. Survival and sex ratios of the intertidal copepod, Tigriopus californicus, following ultraviolet-B (290-320 nm) radiation exposure. Marine Biology, 123(4): 799-804. DOI:10.1007/BF00349123
Smith K C, Macagno E R. 1990. UV photoreceptors in the compound eye of Daphnia magna (Crustacea, Branchiopoda). A fourth spectral class in single ommatidia. Journal of Comparative Physiology A, 166(5): 597-606.
Smith R C, Prézelin B B, Baker K S, Bidigare R R, Boucher N P, Coley T, Karentz D, MacIntyre S, Matlick H, Menzies D. 1992. Ozone depletion:ultraviolet radiation and phytoplankton biology in Antarctic waters. Science, 255(5047): 952-959. DOI:10.1126/science.1546292
Speekmann C L, Bollens S M, Avent S R. 2000. The effect of ultraviolet radiation on the vertical distribution and mortality of estuarine zooplankton. Journal of Plankton Research, 22(12): 2 325-2 350. DOI:10.1093/plankt/22.12.2325
Stearns D E, Forward R B. 1984. Photosensitivity of the calanoid copepod Acartia tonsa. Marine Biology, 82(1): 85-89. DOI:10.1007/BF00392766
Storz U C, Paul R J. 1998. Phototaxis in water fleas (Daphnia magna) is differently influenced by visible and UV light. Journal of Comparative Physiology, 183(6): 709-717. DOI:10.1007/s003590050293
Strom S L. 2001. Light-aided digestion, grazing and growth in herbivorous protists. Aquatic Microbial Ecology, 23: 253-261. DOI:10.3354/ame023253
Sun S, Wang R, Zhang G T, Yang B, Ji P, Zhang F. 2002. A preliminary study on the over-summer strategy of Calanus sinicus in the Yellow Sea. Oceanol. Limnol. Sin., special issue: Zooplankton Population Dynamics: 92-99. (in Chinese with English Abstract)
Sunda W G, Huntsman S A. 1997. Interrelated influence of iron, light and cell size on marine phytoplankton growth. Nature, 390(6658): 389-392. DOI:10.1038/37093
Sweatt A J, Forward R. 1985. Spectral sensitivity of the chaetognath Sagitta hispida Conant. Biological Bulletin, 168(1): 32-38. DOI:10.2307/1541171
Tao Z C, Zhang W C, Sun S. 2004. The wave-specific influence of visible and ultraviolet light on zooplankton behaviour. Marine Sciences, 28(9): 56-61. (in Chinese with English abstract)
Uye S, Huang C, Onbe T. 1990. Ontogenetic diel vertical migration of the planktonic copepod Calanussinicus in the Inland Sea of Japan. Marine Biology, 104(3): 389-396. DOI:10.1007/BF01314341
Uye S, Huang C, Onbe T. 1992. Ontogenetic diel vertical migration of the planktonic copepod Calanus sinicus in the Inland Sea of Japan. Marine Biology, 113(3): 391-400. DOI:10.1007/BF00349164
Uye S. 1986. Impact of copepod grazing on the red-tide flagellate Chattonella antiqua. Marine Biology, 92(1): 35-43. DOI:10.1007/BF00392743
Vallina S M, Simó R. 2007. Strong relationship between DMS and the solar radiation dose over the global surface ocean. Science, 315(5811): 506-508. DOI:10.1126/science.1133680
Wang R, Li C L, Wang K, Zhang W C. 1998. Feeding activities of zooplankton in the Bohai Sea. Fisheries Oceanography, 7(3-4): 265-271.
Wang R, Zuo T, Wang K. 2003. The Yellow Sea cold bottom water-an oversummering site for Calanus sinicus(Copepoda, Crustacea). Journal of Plankton Research, 25(2): 169-183. DOI:10.1093/plankt/25.2.169
Wang S W, Li C L, Sun S, Ning X R, Zhang W P. 2009. Spring and autumn reproduction of Calanus sinicus in the Yellow Sea. Marine Ecology Progress, 379(1): 123-133.
Wang X M, Tang J W, Song Q J, Ding J, Ma C F. 2006. The statistic inversion algorithms and spectral relations of total absorption coefficients for the Huanghai Sea and the East China Sea. Oceanologia et Limnologia Sinica, 37(3): 256-263. (in Chinese with English abstract)
Williamson C E, Zagarese H E, Schulze P C, Hargreaves B R, Seva J. 1994. The impact of short-term exposure to UV-B radiation on zooplankton communities in north temperate lakes. Journal of Plankton Research, 16(3): 205-218. DOI:10.1093/plankt/16.3.205
Zagarese H E, Feldman M, Williamson C E. 1997. UV-B-induced damage and photoreactivation in three species of Boeckella (Copepoda, Calanoida). Journal of Plankton Research, 19(3): 357-367. DOI:10.1093/plankt/19.3.357
Zhang F, Sun S, Zhang G T. 2002a. Preliminary study on egg-laying and hatching of Calanus sinicus (Copepoda: Calanoida) in the laboratory. Oceanol. Limnol. Sin., special issue: Zooplankton Population Dynamics: 10-18.(in Chinese with English abstract)
Zhang G T, Sun S, Sun S. 2002b. Effects of diel spawning rhythm and temperature on egg production and hatching success on Calanus sinicus. Oceanol. Limnol. Sin., special issue: Zooplankton Population Dynamics: 71-77. (in Chinese with English abstract)
Zhang G T, Sun S, Zhang F. 2005. Seasonal variation of reproduction rates and body size of Calanus sinicus in the Southern Yellow Sea, China. Journal of Plankton Research, 27(2): 135-143.
Zhang W C, Wang R. 2000. Effect of concentration of food particles on the feeding behavior of the marine planktonic copepod Calanus sinicus. Acta Oceanologica Sinica, 22(6): 88-94. (in Chinese with English abstract)
Zheng X Y, Zheng Z. 1989. Study on the relationship between mandibular edge and feeding mechanism of Copepoda. Oceanologia et Limnologia Sinica, 20(4): 308-313. (in Chinese with English abstract)
Zuo T, Wang R, Wang K, Gao S W. 2004. Vertical distribution and diurnal migration of zooplankton in the southern Yellow Sea in summer. Acta Ecologica Sinica, 24(3): 524-530. (in Chinese with English abstract)