Effects of temperature regime and food supply on ... - Springer Link

Hydrobiologia (2015) 754:201–214 DOI 10.1007/s10750-015-2279-0

CHINA JELLYFISH PROJECT

Effects of temperature regime and food supply on asexual reproduction in Cyanea nozakii and Nemopilema nomurai Song Feng . Guang-Tao Zhang . Song Sun . Fang Zhang . Shi-Wei Wang . Meng-Tan Liu

Received: 28 January 2015 / Revised: 5 April 2015 / Accepted: 6 April 2015 / Published online: 1 May 2015 Ó Springer International Publishing Switzerland 2015

Abstract Cyanea nozakii and Nemopilema nomurai are two major giant jellyfish species frequently blooming in East Asian waters in recent decades. In order to inspect their asexual reproduction performance in climate regimes associated with global warming, variations of podocyst reproduction and strobilation were investigated at five different temperature regimes (approximate simulation of summer in warm years and cold years: temperature increased

Guang-Tao Zhang and Song Sun contributed equally to this work and should be considered as co-corresponding authors. Guest editors: Song Sun, Xiaoxia Sun & Ian Jenkinson / Giant Jellyfish Blooms and Ecosystem Change S. Feng S. Sun F. Zhang Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China e-mail: [email protected] S. Feng University of Chinese Academy of Sciences, 100049 Beijing, China G.-T. Zhang S. Sun (&) S.-W. Wang M.-T. Liu Jiaozhou Bay Marine Ecosystem Research Station, Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China e-mail: [email protected]

from 18 to 25°C and then decreased to 18 and 10°C in three months, respectively; approximate simulation of autumn in warm years and cold years: temperature decreased from 18 to 10°C in two and three months, respectively; control: constant 18°C), and three food frequencies (once per 3d, once per 6d, and unfed). Strobilation in C. nozakii occurred at warmer temperature (C18°C) but lower temperature (B18°C) in N. nomurai. More than 90% of ephyrae of C. nozakii and N. nomurai were released in the thermal ranges of 22–25°C and 10–13°C, respectively. Higher percentages of polyps produced podocysts, and increased podocyst production was observed at warmer temperature ([18°C) and more food supplies in both species. The numbers of ephyrae in C. nozakii and podocysts in N. nomurai were significantly increased by prolonged duration of 18–25°C in summer during warm years. Simultaneously, prolonged duration of 10–18°C significantly increased strobilation percentage, ephyra, and podocyst production in N. nomurai in the autumn. More polyps of N. nomurai strobilating in the autumn proved adverse to outbreaks of medusae in the following summer. The results of this study indicate that the response of asexual reproduction to ocean warming appears species specific. Increased ephyra production in C. nozakii but higher podocyst reproduction in N. nomurai is expected in summer and autumn during warm years. Keywords Jellyfish bloom Podocyst reproduction Strobilation Ephyra production Global warming

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Introduction Jellyfish blooms have been reported frequently worldwide in recent decades. Although the paradigm that jellyfish are increasing globally (Pauly et al., 1998; Brotz et al., 2012) has been debated (Condon et al., 2013), increases have been reported in specific regions such as the Bering Sea, Black Sea, South American Pacific, and Atlantic coasts (Kideys, 2002; Brodeur et al., 2002; Decker et al., 2014; Mianzan et al., 2014). Frequent outbreaks of the giant jellyfish Cyanea nozakii Kishinouye and Nemopilema nomurai Kishinouye have occurred in the East China Sea, Yellow Sea, and Bohai Sea since 2002, with the most massive blooms of C. nozakii in 2004 and annual blooms of N. nomurai except in 2008, 2010, and 2011 (Ding & Cheng, 2005; Dong et al., 2006; Kawahara et al., 2006; Ding & Cheng, 2007; Uye, 2008; Zhang et al., 2012; Wang et al., 2013; Xu et al., 2013; Sun et al., 2014). C. nozakii is a warm-water species, mainly distributed in the inshore areas of the East China Sea and Liaodong Bay in the Bohai Sea (Dong et al., 2006; Zhang et al., 2012). In contrast, N. nomurai is a eurythermal species that prefers cold water and blooms in the northern East China Sea, Yellow Sea, and Bohai Sea, with a 30˚N distribution boundary caused by the Taiwan Current and other branches of the Kuroshio Current, the south-eastward current off the Changjiang River (Zhang et al., 2012; Sun et al., 2015). These outbreaks of jellyfish pose threats to tourism, fisheries, and coastal facilities such as power plants including nuclear power stations and might be linked to climate change, overfishing, eutrophication, agriculture, or habitat modification arising from human disturbances along the coast (Purcell, 2005; Purcell et al., 2007; Uye, 2008; Richardson et al., 2009; Uye, 2011; Purcell, 2012). In the life cycle of scyphozoan species, the population dynamics of benthic polyps determine the outbreaks of medusae in the summer (Arial, 2009; Lucas et al., 2012; Purcell, 2012). Asexual reproduction of polyps includes podocyst reproduction and strobilation (Kawahara et al., 2006, 2013; Sun et al., 2013; Thein et al., 2013; Sun et al., 2014; Feng et al., 2015). The production and excystment of podocysts contribute to the conservation and increase of polyp population. Ephyrae are liberated via strobilation and then develop into medusae in the summer. As one of the most important human stresses, global warming may increase the population of

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Hydrobiologia (2015) 754:201–214

jellyfish and change the timing and length of their seasons and distributions (Purcell, 2012). Various studies have reported positive correlations between jellyfish abundance and water temperature (reviewed in Purcell et al., 2007; Purcell, 2012). Greater abundance of Chrysaora quinquecirrha in Chesapeake Bay was recorded in warm conditions (Decker et al., 2007). Aurelia aurita and Cyanea spp. in the southern part of the North Sea and in the Irish Sea were more abundant during warm years (Lynam et al., 2010, 2011). High Chrysaora plocamia biomass was associated with El Nin˜o events occurring in warm ‘‘El Viejo’’ regimes (Mianzan et al. 2014). Population increase in jellyfish may occur by asexual reproduction involving increased buds or podocysts of polyps and multiple strobilation. Previous studies of the benthic polyp stages of C. nozakii and N. nomurai primarily sought to determine the thermal thresholds for strobilation and podocyst reproduction via laboratory experiments at constant temperatures. For instance, podocyst proliferation was observed with increasing temperature in these both species (Kawahara et al., 2013; Sun et al., 2013; Thein et al., 2013; Sun et al., 2014; Feng et al., 2015). However, the promotional effect of changed thermal regimes in warm years on asexual reproduction remains unclear. Furthermore, whether or not the responses differ between C. nozakii and N. nomurai is also unknown. Therefore, we hypothesized that prolonging favored thermal regimes in warm years could affect the asexual reproduction strategies of C. nozakii and N. nomurai differently. In this study, we quantitatively assessed strobilation percentage, strobilation duration, percentage of polyps producing podocysts, and ephyra and podocyst production at different temperature regimes and food supplies. The study aimed to compare the responses to ocean warming in terms of asexual reproduction between these two species and deepen understanding of the mechanisms of jellyfish blooms in the coastal seas of China.

Materials and methods Stock culture of polyps Mature medusae of C. nozakii and N. nomurai (six females and four males of each species) were captured near the shore of Jiaozhou Bay in September 2013 and


severally transferred to two 30 m3 ponds in the laboratory at the Institute of Oceanology, Chinese Academy of Sciences, Qingdao. The water temperature and salinity were maintained at 20 ± 0.5°C and 30 ± 0.5 psu, respectively. Adults were removed after planulae were detected. Millions of planulae were produced sexually, attached onto polyethylene plates, and metamorphosed into polyps with two to four tentacles within a week. Polyps were fed with Artemia nauplii for 1–2 h every 3 days. The water in the pond was then replaced with freshly filtered seawater (dissolved oxygen [7 mg L-1). One month later, 16-tentacle polyps had developed fully, while the water temperature and salinity were 18 ± 0.5°C and 30 ± 0.5 psu, respectively. Finally, a stock population of polyps was cultivated at a water temperature and salinity of 18 ± 0.5°C and 30 ± 0.5 psu, respectively. Experimental design According to annual and inter-annual variation in seawater temperature in Jiaozhou Bay, Yellow Sea of China, the favorable temperature regimes in summer and autumn during warm years and cold years were approximately simulated in the experiment, respectively. Five treatments were conducted in different incubators as follows (Fig. 1). Treatments I and II approximately simulated the temperature regimes in the summer during warm and cold years, respectively. Treatment I: the temperature initially increased from 18°C to 25°C in 1°C increments every 4 days and then decreased again from 25 to 18°C in 1°C reductions every 9 days. Treatment II: the temperature was elevated to 25°C as in Treatment I but then dropped from 25 to 10°C in 1°C reductions every 4 days. Treatment III: control group in which the temperature was maintained at 18°C for 100 days. Treatment IV and V were designed to compare the autumn conditions in warm and cold years, with the temperature reduced from 18 to 10°C in 1°C reductions every 11 and 8 days in Treatment IV and V, respectively. Three different feeding frequencies were also conducted: once per 3 days (3d), once per 6 days (6d), and unfed. Well-stretched 16-tentacle polyps with similar calyx diameters (mean: 695.4 ± 12.7 lm) were selected for the experiments. The polyethylene plates to which the polyps had become attached were cut into pieces of 2 9 1 cm. On each piece of plate lived one polyp, which was cultivated in a 50-mL bowl. The polyp was

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Fig. 1 Schematic representation of the experimental design. According to annual and inter-annual variations in seawater temperatures in Jiaozhou Bay, Yellow Sea of China, favorable temperature regimes for asexual reproduction in summer and autumn may last for 2 or 3 months during cold and warm years, respectively. The experiment was designed as five different temperature regimes. Treatments I and II approximately simulated the temperature regimes in the summer during the warm and cold years, respectively. The temperature was first increased from 18 to 25°C in 1°C increments every 4 days and then was decreased from 25 to 18°C in 1°C reductions every 9 days in Treatment I. In Treatment II, the temperature was first elevated to 25°C as in Treatment I but then dropped from 25 to 10 in 1°C reductions every 4 days. Treatment III was a control group, in which the temperature was maintained at 18°C for 100 days. Treatments IV and V approximately compared temperature regimes of autumn in warm and cold years. The temperature was decreased from 18 to 10 in 1°C reductions every 11 or 8 days in Treatments IV and V, respectively

in an upright position from the bottom in the bowl. Ten Artemia nauplii were added into the bowl with a pipette and randomly preyed on. Uneaten food was removed with a pipette after 1–2 h, and the seawater in the bowl was replaced with fresh, filtered seawater of the same temperature and salinity. Each combination of temperature regime and food supply comprised three replicates, and each replicate contained 10 polyps. The experiment started on November 16, 2013. The numbers of strobilae, polyps producing podocysts in every replicate, and podocysts per polyp were recorded under a dissecting microscope (Olympus SZ61) every 2 days. The number of ephyrae per polyp was counted and removed with a pipette every 2 days during strobilation. Polyps that developed by excystment were excised with dissecting needles and were not considered in the experiment. To elaborate the strobilation time in detail, the stage from polyps to the first fully developed strobila when the experiment started was defined as the Pre-Strobilation Stage (PS).

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The fully developed strobilae appeared when the tentacles of polyps had completely regressed, all the discs had developed, and ephyrae started to pulsate rhythmically. The Strobilation Interval Stage (SI) was the period from the first strobila to the first ephyra released. Strobilation Stage (SS) was from the first to last ephyra released. The time for microscopic observation of every bowl took less than 1 min. Calculations and statistical analysis Strobilation percentage or percentage of polyps producing podocysts was calculated for each replicate based on the total numbers of strobilae or polyps producing podocysts, divided by the numbers of polyps in each replicate. Ephyra and podocyst production was represented by the average values of the total numbers of ephyrae and podocysts produced per polyp after the experiment finished, respectively. Strobilation duration (PS, SI, SS) was calculated as the average durations of PS, SI, SS in three replicates, respectively. Three null hypotheses were examined. The first null hypothesis H01 was that asexual reproduction of C. nozakii and N. nomurai had no difference in strobilation percentage, strobilation duration, and percentage of polyps producing podocysts, ephyra, and podocyst production. The second null hypothesis H02 was that temperature regime did not affect the strobilation percentage, strobilation duration (PS, SI, SS), percentage of polyps producing podocysts, ephyra, or podocyst production in either C. nozakii or N. nomurai. The third null hypothesis H03 was that strobilation and podocyst reproduction was independent of food supply. H01, H02, and H03 were analyzed with three-way ANOVAs after testing for normality and equality in variance of the data (SPSS 16.0). Percentages were arcsine square root transformed before statistical analysis. If the overall ANOVA results were significant, Bonferroni was performed to test among experimental combinations.


differed significantly among species, temperature regimes, and food supply. H01, H02, and H03 were thus rejected (Table 1). The interactions among three factors also remarkably affected strobilation percentage. For C. nozakii polyps, strobilation occurred in Treatment I, II, and III, with no significant differences between Treatment I and II (Bonferroni, P = 1.000). Warmer temperatures ([18°C) favored strobilation. The highest strobilation percentage occurred at the combination of Treatment II and 6d feeding (97.0 ± 5.2%) (Fig. 2). However, polyps of N. nomurai strobilated in Treatment II, III, IV, and V, with significant differences between Treatment IV and V (Bonferroni, P = 0.001). Lower temperature (\18°C) was crucial to strobilation. And strobilation percentage was higher with prolonged favored duration of 10–18°C. The highest strobilation percentage appeared at Treatment IV 6d feeding group (76.4 ± 16.6%) and the lowest at Treatment II 3d feeding group (2.4 ± 4.1%) (Fig. 2). No strobilation occurred in Treatment I. Strobilation percentage was significantly lower in the unfed group than two fed groups for C. nozakii and N. nomurai. However, no significant difference was tested between 3d and 6d feeding groups (Bonferroni, P = 1.000). Besides, in C. nozakii polyps, there appeared repeated strobilations in the experiment but not in N. nomurai polyps. Strobilation was observed at most five times in Treatment I 6d feeding group for C. nozakii polyps. Polyps strobilated just once or twice in unfed groups. As the frequencies of strobilation increased, strobilation percentage significantly decreased. Strobilation percentage declined from 92.3% of first strobilation to 5.0% of fourth strobilation and from 92.3% of first strobilation to 2.7% of fifth strobilation in Treatment I 3d and 6d groups, respectively. Similarly, it decreased from 83.5% of first strobilation to 2.6% of fourth strobilation and from 97.0% of first strobilation to 2.8% of third strobilation in Treatment II 3d and 6d groups, respectively (Fig. 3).

Results Strobilation duration Strobilation Strobilation percentage All C. nozakii and N. nomurai polyps survived during the experimental period. The strobilation percentage

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PS and SI were significantly different between species, but not SS. Temperature regimes had a significant effect on PS, SI, and SS. However, food supply only prominently affected the SS (Table 2). The longest PS and SS of C. nozakii and N. nomurai were present at

Hydrobiologia (2015) 754:201–214 Table 1 Summary of three-way ANOVA results among species, temperature regime, and food supply on strobilation percentage, percentage of polyps producing podocysts, ephyra, and podocyst production

205

SS

df

MS

F

P

Strobilation percentage Species

0.154

1

0.154

8.874

Temperature regime

1.797

4

0.449

25.906

0.000

Food supply

1.400

2

0.700

40.362

0.000

15.779

4

3.945

227.501

0.000

Species 9 Temperature regime

0.004

Species 9 Food supply

0.164

2

0.082

4.734

0.012

Temperature regime 9 Food supply

0.356

8

0.045

2.567

0.018

Species 9 Temperature regime 9 Food supply Error

1.704 1.040

8 60

0.213 0.017

12.281

0.000

37.935

90

Species

0.253

1

0.253

6.377

0.014

Temperature regime

3.074

4

0.769

19.364

0.000

Food supply

5.039

2

2.519

63.472

0.000

Total Ephyra production

Species 9 Temperature regime Species 9 Food supply

21.392

4

5.348

134.730

0.000

0.050

2

0.025

0.634

0.534


1.235

8

0.154

3.888

0.001

Species 9 Temperature regime 9 Food supply

6.333

8

0.792

19.942

0.000

Error

2.382

60

0.040

Total

58.710

90

Percentage of polyps producing podocysts Species

10.061

1

10.061

376.298

0.000

Temperature regime Food supply

3.619 6.198

4 2

0.905 3.099

33.838 115.921

0.000 0.000


0.483

4

0.121

4.513

0.003


1.856

2

0.928

34.704

0.000


0.985

8

0.123

4.603

0.000


0.827

8

0.103

3.866

0.001

Error

1.604

60

0.027

Total

60.851

90

155.558

1

609.451

0.000

Podocyst production Species

Percentages were arcsine square root transformed before statistical analysis SS sum of square, MS mean square, df Degree of freedom

155.558

Temperature regime

74.425

4

18.606

72.896

0.000

Food supply

90.168

2

45.084

176.632

0.000 0.000


61.547

4

15.387

60.283


77.756

2

38.878

152.318

0.000


37.875

8

4.734

18.548

0.000

Species 9 Temperature regime 9 Food supply Error

34.870 15.315

8 60

4.359 0.255

17.077

0.000

742.338

90

Total

Treatment III, 6d feeding group (80 ± 18d), Treatment I, 3d feeding group (55 ± 22.1d) and Treatment II, 6d feeding group (100d), Treatment IV, 3d feeding group (55 ± 26.9d), respectively (Fig. 4). SS was

significantly longer in Treatment I than Treatment II for C. nozakii (Bonferroni, P = 0.001) and in Treatment IV than Treatment V for N. nomurai (Bonferroni, P = 0.003). SS was therefore prolonged with longer

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206


Treatment I than in Treatment II (Bonferroni, P = 0.000) and by N. nomurai polyps in Treatment IV than in Treatment V (Bonferroni, P = 0.001). Prolongation of the favored duration of strobilation notably enhanced ephyra production in both C. nozakii and N. nomurai. Significantly fewer ephyrae were released in the unfed groups. But ephyra production between the 3d and 6d feeding groups did not remarkably differ (Bonferroni, P = 0.822). Podocyst production

Fig. 2 Mean strobilation percentages for Cyanea nozakii and Nemopilema nomurai polyps at five temperature regimes (Treatments I, II, III, IV, and V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed)

favored duration of strobilation in C. nozakii and N. nomurai. SS was markedly shorter in unfed groups compared with fed groups, with no significant difference between the 3d and 6d feeding groups (Bonferroni, P = 1.000). Ephyra production In our experiment, only one disc was formed by 99.3% strobilae and two discs by 0.7% strobilae for C. nozakii. However, 1-4 discs were developed by a N. nomurai strobila. During strobilation, ephyrae were rapidly liberated during a short period in both C. nozakii and N. nomurai. More than 90% of all ephyrae were released within 25–40 days and after 60 days, when the temperatures ranged from 22 to 25°C and from 10 to 13°C, respectively (Fig. 5). Species, temperature regime, and feeding frequency all significantly affected ephyra production. H01, H02, and H03 were therefore rejected (Table 1). Besides, there was also marked influence of interactions among three factors, except for the two factors, species, and food supply. The maximum numbers of C. nozakii and N. nomurai ephyrae appeared at the combination of Treatment I, 3d or 6d feeding (2.0 ± 0.2 ephyrae polyp-1) and Treatment IV, 6d feeding (1.6 ± 0.3 ephyrae polyp-1), followed by Treatment II, 3d feeding (1.5 ± 0.1 ephyrae polyp-1) and Treatment III, 3d feeding (1.3 ± 0.6 ephyrae polyp-1), respectively (Fig. 5). No C. nozakii ephyrae were released in Treatment IV or V and no N. nomurai ephyrae in Treatment I or II. Significantly more ephyrae were produced by C. nozakii polyps in

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There were significant differences in percentage of polyps producing podocysts among the two species, the temperature regime, feeding frequency, and their interactions (Table 1). A higher percentage of polyps produced podocysts at warmer temperature ([18°C) and at higher feeding frequencies. In C. nozakii, a maximum of 36.4% of polyps produced podocysts, compared with all N. nomurai polyps in Treatment I, II, and III (Fig. 6). In Treatments I and II, the numbers of podocysts increased steeply before the temperature declined to 18°C in both C. nozakii and N. nomurai, whereas podocysts were not produced below 16°C in C. nozakii or below 13°C in N. nomurai in Treatment IV and V (Fig. 7). Podocyst production was significantly affected by species, temperature regime, feeding frequency, and their interactions. H01, H02, and H03 were thus rejected (Table 1). The maximum podocyst production of C. nozakii and N. nomurai polyps occurred at Treatment II 6d feeding group (0.39 ± 0.05 podocysts polyp-1) and Treatment I 3d feeding group (9.1 ± 0.6 podocysts polyp-1), respectively (Fig. 7). For C. nozakii polyps, there were no significant differences in podocyst production between Treatments I and II (Bonferroni, P = 1.000) or between Treatments IV and V (Bonferroni, P = 1.000). However, N. nomurai polyps produced significantly more podocysts in Treatment I than in Treatment II (Bonferroni, P = 0.007) and in Treatment IV than in Treatment V (Bonferroni, P = 0.040). Prolongation of the favored duration in warm years was therefore conductive to podocyst reproduction in N. nomurai. Higher podocyst production was associated with increased food supply; however, there was no significant difference in podocyst production between 3d and 6d feeding frequencies (Bonferroni, P = 1.000) for C. nozakii, though significantly fewer podocysts were produced in the unfed groups.


207

Fig. 3 Changes in the cumulative percentage of strobilation for C. nozakii at five temperature regimes (Treatments I, II, III, IV, V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed). No strobilation occurred in Treatment IV and V, which are not shown in figure

Discussion Effects of temperature regime on asexual reproduction in C. nozakii and N. nomurai Strobilation The strobilation percentage significantly differed among temperature regimes in both C. nozakii and N. nomurai. Strobilation of C. nozakii polyps occurred as the temperature increased from 18 to 25°C, then decreased to 18°C, but not when the temperature dropped from 18 to 10°C, over either 2 or 3 months. This was consistent with the study of Sun et al. (2013), who found that strobilation occurred at higher

temperatures (21.5–25°C). Warmer temperature contributed to the higher strobilation percentage in C. nozakii, though our experiment also showed that its polyps could strobilate at 18°C after 80 days. The experiment also suggested that C. nozakii polyps would not strobilate in autumn at temperatures below 18°C during cold or warm years in coastal sea of China, according to the results of Treatments IV and V. In contrast, N. nomurai polyps strobilated as the temperature fell from 18 to 10°C over either 2 or 3 months or at a constant 18°C, but no strobilation occurred at higher temperatures (increase from 18 to 25°C and then decrease to 18°C in 2 or 3 months). This is in agreement with Kawahara et al. (2013), who

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208


Table 2 Summary of three-way ANOVA results among species, temperature regime, and food supply on strobilation duration including pre-strobilation, strobilation interval stage and strobilation stage SS

df

MS

F

P

Pre-strobilation stage Species

15418.711

1

15418.711

105.592

0.000

Temperature regime

10259.489

4

2564.872

17.565

0.000 0.388

Food supply Species 9 Temperature regime Species 9 Food supply

281.156

2

140.578

0.963

22418.956

4

5604.739

38.383

0.000

3845.956

2

1922.978

13.169

0.000


15679.844

8

1959.981

13.422

0.000


15221.711

8

1902.714

13.030

0.000

146.022

0.000

Error

8761.333

60

Total

166652.000

90

Species

18.678

1

18.678

21.278

Temperature regime

23.444

4

5.861

6.677

0.000

4.956

2

2.478

2.823

0.067

88.156 1.089

4 2

22.039 0.544

25.108 0.620

0.000 0.541

Strobilation interval stage

Food supply Species 9 Temperature regime Species 9 Food supply

5.489

8

0.686

0.782

0.620



10.244

8

1.281

1.459

0.192

Error

52.667

60

0.878

Total

305.000

90

26.678

1

0.185

0.669

Strobilation stage Species

26.678

Temperature regime

3095.489

4

773.872

5.367

0.001

Food supply

3406.822

2

1703.411

11.813

0.000


19500.600

4

4875.150

33.808

0.000


213.089

2

106.544

0.739

0.482


499.178

8

62.397

0.433

0.897

2.758

0.012


3181.133

8

397.642

Error

8652.000

60

144.200

Total

56751.000

90

SS sum of square, MS mean square, df Degree of freedom

showed that N. nomurai polyps could strobilate at 11–15°C by cooling from 19°C. In our previous study, we suggested that strobilation could occur at 10–19°C in spring (Feng et al., 2015). However, the current study indicates that strobilation was likely during autumn cooling from 18 to 10°C, though N. nomurai ephyrae and juvenile medusa have not been investigated in situ in late autumn and early winter (November and December) in the northern East China sea, Yellow Sea, or Bohai Sea. Higher strobilation percentage might be expected in autumn during warm

123

years because of the longer favored duration of 10–18°C. Strobilation duration PS, SI, and SS were markedly impacted by temperature regime in C. nozakii and N. nomurai polyps. Longer duration of temperature regime of favorable strobilation significantly prolongs SS, but not PS or SI, in both species. The prolonged SS results in C. nozakii and N. nomurai polyps liberating more ephyrae in


209

Fig. 4 Mean strobilation durations (PS, SI, SS) of Cyanea nozakii and Nemopilema nomurai polyps at five temperature regimes (Treatments I, II, III, IV, V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed). The stage from polyp to the first fully developed strobila was defined as the prestrobilation stage (PS). The fully developed strobilae appeared when the tentacles of polyps had absolutely regressed, all the discs had developed, and ephyrae started to pulsate rhythmically. The strobilation interval stage (SI) was the period from the first strobila to the release of the first ephyra, and the strobilation stage (SS) was the period from the release of the first to the last ephyra

Fig. 5 Changes in cumulative numbers of ephyrae per polyp for Cyanea nozakii and Nemopilema nomurai at five temperature regimes (Treatments I, II, III, IV, V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed)

summer and autumn during warm years. Our study has revealed that the strobilae of C. nozakii developed more rapidly at higher temperature, which is compatible with the study of Sun et al. (2013), who found that strobilation appeared 10 days earlier at 25°C than at 21.5°C.

Kawahara et al. (2013) showed that strobilation of N. nomurai polyps occurred at 11–15°C within one month; however, strobilation occurred at least 40 days later in the present study. This difference in PS may be the result of different experimental methods. Kawahara et al. (2013) maintained polyps at different

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Fig. 6 Mean percentages of polyps producing podocysts (mean±SD) for Cyanea nozakii and Nemopilema nomurai at five temperature regimes (Treatments I, II, III, IV, V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed)

temperatures by rapid cooling from 19°C, while our experiments approximately simulated the autumn cooling process in cold and warm years. In addition, we previously concluded that strobilation commenced after a lag time of 1 month at most for over-wintering N. nomurai polyps (shorter PS with longer durations at low temperature in winter), when temperature rose in spring (Feng et al., 2015). Strobilation of N. nomurai polyps in autumn thus appeared to require a longer run-up time than in spring. Ephyra Ephyra production remarkably differed among temperature regimes in C. nozakii and N. nomurai. More ephyrae were liberated at 22–25°C and 10–13°C in C. nozakii and in N. nomurai, respectively, as shown in previous studies (Sun et al., 2013; Feng et al., 2015). Zhang et al. (2012) suggested that C. nozakii and N. nomurai may be respectively considered as warmwater and cold-water preferring species, in agreement with the results of the present study. Moreover, prolonged favorable duration of strobilation in warm years significantly increased ephyra production in both species. In C. nozakii, only one ephyra was developed per strobila in most cases (two ephyrae were formed by a strobila in one case). But repeated strobilations were observed in our experiment, up to five times at most. As in Aurelia labiata in Washington State, USA (Purcell, 2007), strobilation percentage gradually decreased with the increasing strobilation frequencies. Accordingly, the

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increase in ephyra production in warm years may be attributable to the multiplication of strobilation times resulting from the prolonged strobilation stage. The percentage of polyps strobilating twice and three times significantly increased in warm years above 18°C (Treatment I 3d feeding: 66.7% 6d feeding: 67.8%), and most polyps only strobilated once in cold years above 18°C (Treatment II 3d feeding: 47.2%, 6d feeding: 57.3%) because of the shorter duration of favored strobilation (Fig. 8). In this study, N. nomurai polyps formed one to four ephyrae per strobila, as a polydisc, consistent with the report of Feng et al. (2015). The increased ephyra production in warm years resulted from the higher strobilation percentage at the prolonged duration of favored strobilation. More polyps would strobilate at 10–18°C in the autumn during warm years. However, we found that 8% of polyps were lost and 92% degenerated into stalks or polyps with fewer than eight tentacles (40–500 lm) after strobilation. All of these hardly grew and developed into 16-tentacled polyps at extremely low temperatures in the winter or during strobilation in the next spring (Sun et al., 2014; Feng et al., 2015). Fewer polyps would thus strobilate in the following spring. Our experiments also showed that only 33% of ephyrae remained alive at 5°C after 2 months, and their total body diameters gradually shrank. The above results demonstrate that ephyrae liberated in the autumn hardly survive at low temperatures in the winter and therefore do not provide a crucial supplement for the N. nomurai medusae in the following summer. Prolonged duration of 10–18°C in the autumn during warm years therefore has a negative effect on the massive outbreaks of N. nomurai in the following summer. Podocyst reproduction The temperature regime had a significant impact on podocyst production in both C. nozakii and N. nomurai. More podocysts were produced at warmer temperatures, consistent with previous reports (Sun et al., 2013; Thein et al., 2013; Sun et al., 2014; Feng et al., 2015). Dong et al. (2008) described that asexual reproduction in C. nozakii polyps included podocysts and stoloncysts (cysts developed at the distal end of an extended stolon). However, only two stoloncysts were observed in our experiments, suggesting that podocysts represented the dominant asexual reproduction strategy in C. nozakii. Podocyst production was not


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Fig. 7 Changes in cumulative podocysts per polyp for Cyanea nozakii and Nemopilema nomurai at five temperature regimes (Treatments I, II, III, IV, V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed)

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Fig. 8 Mean percentages of strobilation times for Cyanea nozakii polyps at five temperature regimes (Treatments I, II, III, IV, V) and three feeding frequencies (once per 3d (3d), once per 6d (6d), unfed) Strobilation did not occur in Treatments IV and V (not shown)

markedly increased by prolonged favored duration in warm years, but ephyra production was increased. This suggests that polyps of C. nozakii tended to strobilation rather than podocyst reproduction in the summer during warm years. In contrast, longer favored duration in warm years significantly increased podocyst production in N. nomurai polyps. Higher podocyst production occurred before the seawater temperature decreased to 18°C in the summer during warm years. Kawahara et al. (2013) suggested that the excystment of accumulated podocysts in favorable conditions is conducive to jellyfish blooms. Therefore, the accumulation of higher podocyst production in warm years may crucially determine the polyp population size and may lead to N. nomurai blooms in the future. Effect of food supply on asexual reproduction in C. nozakii and N. nomurai Food supply significantly affected strobilation percentage, the percentage of polyps producing podocysts, ephyra, and podocyst production in C. nozakii and N. nomurai polyps, in accord with previous studies (Sun et al., 2013; Thein et al., 2013; Sun et al., 2014). However, there were no remarkable differences in strobilation percentage or ephyra production for C. nozakii and N. nomurai polyps between the 3d and 6d feeding groups. This may be on the one hand because the initial calyx diameters of the polyps, which determined strobilation and ephyra production (Russell, 1970), were similar. On the other hand, it may

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have been because polyps fed on fewer Artemia nauplii during strobilation. More podocysts of N. nomurai were produced at higher feeding frequency. But there was no significant difference in podocyst production between the 3d and 6d feeding groups for C. nozakii polyps. It is possibly that more energy was used for strobilation at warmer temperatures ([18°C), and polyps fed fewer Artemia nauplii at lower temperatures (\18°C). We previously demonstrated that strobilation percentage and ephyra production of N. nomurai polyps in spring were not markedly affected by food supply (Feng et al., 2015). It is therefore concluded that food supply played a key role in the growth of polyps and in ephyra and podocyst production in autumn, as well as podocyst production in summer, but not in strobilation during the spring. Varied responses to global warming in asexual reproduction strategies Temperature regimes might affect long-term variations in jellyfish populations (Purcell, 2005, 2012). Global warming may increase the jellyfish population size (Purcell, 2012). In addition to increased temperature, annual prolongation of favorable duration in warm years may be a crucial driver of recent jellyfish blooms. Our experiments have demonstrated that the response to prolonging favored thermal regimes in warm years in terms of asexual reproduction was species specific. In Jiaozhou Bay, highest abundance of C. nozakii appears in August and September, and this species disappears later than N. nomurai (Wang et al., 2012). Our results strongly suggest that more ephyrae and longer jellyfish duration occurs in summer and autumn during warm years. Podocyst reproduction of C. nozakii was weaker than strobilation in warm years, suggesting that podocyst reproduction may not be the major contribution to C. nozakii blooms. In contrast, podocyst reproduction in N. nomurai increased significantly above 18°C in the summer during warm years, the rate of which was higher than C. nozakii. The accumulation of more podocysts in warm years may contribute to preserve and increase polyp population and then further support subsequent medusa blooms. Our previous study suggested that longer duration at low temperature in winter and a continuous 10–13°C period in spring were conductive to N. nomurai blooms (Feng et al., 2015). In this study, prolonged duration of favored strobilation (10–18°C)


in the autumn proved negative to the medusa blooms in the following summer owing to low winter survival of ephyrae and fewer polyps strobilating in the following spring. Therefore, N. nomurai blooms would be expected when a shorter duration of 10–18°C in autumn and longer duration at low temperature in winter appeared.

Conclusion Global warming may worsen the incidence of recent massive outbreaks of C. nozakii and N. nomurai in East Asian waters. The prolongation of favored thermal regimes in warm years may play a crucial role. The results of this study have demonstrated that the responses of asexual reproduction strategies in C. nozakii and N. nomurai to prolonging favored temperature regimes differ significantly. Prolonged favorable duration of 18–25°C in summer during warm years significantly promotes ephyra production in C. nozakii and podocyst production in N. nomurai. Prolonged favorable duration of 10–18°C in autumn during warm years led to more N. nomurai polyps strobilating, which appears negative to N. nomurai blooms in the following summer. Therefore, prolonging favored temperature regimes in warm years may drive jellyfish blooms with species specificity. Increased ephyra production in C. nozakii but higher podocyst reproduction in N. nomurai is expected in summer and autumn during warm years. Acknowledgments We thank Miss Zhenghua Zhang for caring for the polyps. We are grateful to the fishermen of the ship ‘Lulao Yu 2030’, Qun Liu, Aiyong Wan, and Yantao Wang, for help with sampling Cyanea nozakii and Nemopilema nomurai adults for fertilization in the field. We are deeply thankful to Professor Ian Jenkinson for editing and constructive suggestions on the manuscript. We are grateful to two anonymous referees for suggesting important improvements. This research was supported by National Key Fundamental Developing Project No. 2011CB403601. Song Feng completed the experiment and wrote this manuscript with the help of Guangtao Zhang and Song Sun. Guangtao Zhang and Song Sun contributed equally to this work and should be considered as cocorresponding authors.

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