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Potential grazing intensity directly determines the extent of grazer-induced colony formation in Scenedesmus obliquus
Biochemical Systematics and Ecology 61 (2015) 271e277
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Potential grazing intensity directly determines the extent of grazer-induced colony formation in Scenedesmus obliquus Xuexia Zhu, Haihong Nan, Qinwen Chen, Zhongqiu Wu, Xinyan Wu, Yuan Huang**, Zhou Yang* Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2015 Received in revised form 16 June 2015 Accepted 27 June 2015 Available online 6 July 2015
The genus Scenedesmus can form large colonies to provide protection against predation by herbivores. Scenedesmus has been investigated to elucidate the varying extents of colony formation in response to predation. However, limited information is available regarding the factors directly affecting the extent of colony formation responses. This study investigated the colony formation responses of Scenedesmus obliquus to infochemicals derived from Daphnia of different ages with the same biomass or feeding intensity. Results showed that water containing Daphnia infochemicals can induce the colony formation in S. obliquus regardless of age, biomass, or feeding intensity of Daphnia. The infochemicals derived from 1-day-old Daphnia caused a higher number of cells per particle than those from 5-day-old Daphnia when the grazers were of the same biomass; the infochemicals from 1-day-old Daphnia also maintained the algal colonial morph longer than those from 3-day-old Daphnia. The number of cells per particle did not differ among the algal populations treated with infochemicals from Daphnia of different ages when the feeding intensity of grazers was held constant. Therefore, potential grazing intensity directly determines the extent of the grazer-induced colony formation in S. obliquus. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Colony formation Daphnia magna Feeding intensity Grazer biomass Scenedesmus obliquus
1. Introduction In aquatic ecosystems, phytoplankton are very susceptible to herbivory by zooplankton. Many planktonic organisms sense li, 2010; Van Donk et al., 2011). potential predators by using predator-derived infochemicals (Tang, 2003; Lundgren and Grane Phytoplankton react by exhibiting morphological responses that reduce the predation risk; among these responses, colony formation is interpreted as a defense mechanism against grazing (Jakobsen and Tang, 2002; Kampe et al., 2007; Tang et al., 2008). The green alga Scenedesmus is one of the most investigated phytoplankton in terms of grazer-induced colony formation (Hessen and Van Donk, 1993; Lürling, 2003a; Yang et al., 2007). Scenedesmus responds by forming large colonies, such as an eight-celled colony, when the threat of predation by grazers is high; as a result, a collective size beyond the predation capacity of zooplankton is formed. Herbivorous zooplankton species, such as cladocerans and rotifers, trigger the unicellular colony transformation of Scenedesmus to varying extents (Lampert et al., 1994; Yang et al., 2006).
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Huang), [email protected] (Z. Yang). http://dx.doi.org/10.1016/j.bse.2015.06.035 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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In aquatic ecosystems, algae are not confronted with constant predation pressure, but rather experience rapid variation in grazing pressure. In addition to the taxonomic diversity of predators, the abundance and feeding activity of a specific grazer can vary spatially and temporally (Sterner, 1989; Viejo and Åberg, 2003). Inducible algae are expected to respond proportionally on the basis of the predation risk level because costs are associated with induced defenses (Lürling and Van Donk, 2000; Beardall et al., 2009). For instance, Lampert et al. (1994) and Lürling and Van Donk (1997) investigated the relationship between the number of Daphnia and the subsequent induced colony formation of Scenedesmus. Using Daphnia of the same age, Wu et al. (2013) found that the colony formation of Scenedesmus is dependent on the density of Daphnia; the number of cells per particle increases as the density of Daphnia increases. Zhu et al. (2015) found that large Daphnia causes the formation of high proportions of large Scenedesmus colonies when animals of different sizes were kept at the same density. High density and large body increase grazer biomass; both parameters may be responsible for the strengthened colony formation responses of Scenedesmus. In addition to grazer biomass, grazing pressure is a direct measure of the predation risks that algae face. Grazing pressure likely plays a key role in determining the extent of the grazer-induced colony formation response of Scenedesmus because the grazing activity of zooplankton triggers unicellular colony transformation (Von Elert and Franck, 1999). Lürling (2003a) found that water from cultures with high grazing rates induces the formation of more Scenedesmus obliquus colonies than water from cultures with low grazing rates. However, a high density of animals (leading to higher grazer biomass) has been used in cultures with high grazing rates, which may also contribute to the strengthened colony formation response. To eliminate such uncertainty, we conducted an experiment to evaluate the colony formation response of S. obliquus to infochemicals derived from Daphnia magna of different ages with the same biomass. We hypothesized that Daphnia at the same biomass causes similar extents of inducing colony formation; however, our results rejected this hypothesis. In a second experiment, infochemicals derived from D. magna of different ages with the same feeding intensity were used to induce unicellular colony transformation. Our results confirmed that potential grazing intensity directly determines the extent of colony formation in S. obliquus. 2. Materials and methods 2.1. Algae and zooplankton S. obliquus (FACHB-416) was obtained from the Institute of Hydrobiology, Chinese Academy of Science. The algae were precultured axenically in liquid BG-11 medium (Rippka et al., 1979) at 25 ± 1 C and illuminated at 40 mmol photons m2 s1 by using fluorescent lamps in a lightedark period of 12 h:12 h. The cladoceran D. magna, a laboratory clone maintained for more than 10 years, was cultured in beakers and fed with S. obliquus under the same conditions as those for culturing S. obliquus. 2.2. Experimental protocol Standardized bioassays were performed to determine the morphological responses of Scenedesmus to zooplankton filtrates (Hessen and Van Donk, 1993; Lürling, 2003b; Yang et al., 2007). Two experiments were conducted successively as follows: Experiment 1. To produce the test water containing infochemicals from Daphnia of different ages with the same biomass, 1-, 3- and 5-day-old D. magna were used in the experiment. The Daphnia biomass was quantified as dry weight. After three rinses with redistilled water, Daphnia at each age was dried at 80 C for 48 h, cooled at room temperature, and weighed to determine the average dry weight per age. To ensure that the Daphnia infochemicals can induce colony formation in Scenedesmus, we used Daphnia biomass of 12.8 ± 0.1 mg dry weight L1 (equal to the biomass of 5-day-old Daphnia at a density of 120 inds L1) in the experiment. Different densities of D. magna at each age (Table 1) were selected to reach the same grazer biomass. In each age, D. magna was fed on S. obliquus at a density of 1 106 cells mL1 for 24 h. The actual feeding intensity corresponding to each age of grazers was calculated on the basis of the equation of Frost (1972). To prepare the test water, we filtered the medium through a 60 mm nylon mesh and then through a 0.10 mm rinsed glass fiber filter (Millipore Corporation, USA) to remove the zooplankton and algae, respectively. A suspension culture of S. obliquus that was not exposed to Daphnia was filtered in the same way and used as control. Aliquots of exponentially growing unicellular S. obliquus from the preculture were transferred to 250 mL Erlenmeyer flasks, which were sealed with cellulose plugs. Each batch culture contained 135 mL of unicellular algal inoculum and either 15 mL of control water or 15 mL of test water. Similar initial algal concentrations were prepared for each batch culture with a
Table 1 Body size, dry weight, and density of Daphnia magna of different ages in experiment 1. Age (day)
Body size (mm)
Dry weight (mg ind1)
Density (inds L1)
1 3 5
0.788 ± 0.006 1.299 ± 0.013 2.313 ± 0.030
0.008 0.046 0.106
1590 280 120
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density of 2.0 104 cells mL1. The control group and three different treatments (containing 1-, 3-, and 5-day-old D. magnacultured water, respectively) were run in six replicates for eight days under the previously described culture conditions. The flasks were shaken thrice daily and rearranged randomly to avoid the effect of minor differences in illumination. Experiment 2. Two-, 4- and 6-day-old D. magna were used in experiment 2; the body size for each age was determined by measuring at least 30 individuals. According to the equation of Frost (1972), we obtained a series of feeding intensities corresponding to different densities of Daphnia at each age in a pre-experiment. Different numbers of Daphnia at each age were selected on the basis of these data to achieve the same feeding intensity (Table 2 for the densities and actual feeding intensities). D. magna was then fed on S. obliquus to prepare the test water, which was used to induce the colony formation of Scenedesmus in accordance with the procedures performed in experiment 1. 2.3. Algal growth and colony formation Samples (2 mL) were collected daily and preserved in Lugol's solution (2%). The abundance and the number of algal cells in different morphological particles (single cells; two-, four-, eight-celled colonies; and other colonies) were counted using a hemocytometer (Tianlong XB-K-25, China) with an optical microscope (Olympus 6V20WHAL, Japan). The growth rates were determined as the slope of the ln of cell abundance versus time from daily samples (Yang et al., 2010). The mean proportion of cells in different particles and the mean number of cells per particle were calculated from the counts. 2.4. Statistical analyses Data were presented as mean ± SE. The growth rates and feeding intensities were compared through one-way ANOVA with Daphnia ages as the factor. Differences in the number of cells per particle between groups were compared through repeated measures ANOVA. The differences obtained on each day were analyzed through multivariate analysis of variance with the Daphnia test water as the factor. Significantly different means were distinguished by a Holm-Sidak post hoc comparison test (P < 0.05). Statistical analyses were performed using SPSS 16.0 software. 3. Results 3.1. Experiment 1 During the test water preparation, the feeding intensities of 1-, 3- and 5-day-old Daphnia were 55.17 ± 5.60, 41.67 ± 1.56, and 31.01 ± 2.33 106 cells day1, respectively (Fig. 1). One-way ANOVA indicated significant differences in feeding intensities (F ¼ 11.184, P ¼ 0.009). After Daphnia-cultured water was added to the algal medium, the algal growth was comparable to that cultured in the control water. The growth rates of S. obliquus exposed to infochemicals from 1-, 3-, and 5-day-old Daphnia at the same biomass were 0.86 ± 0.11, 0.84 ± 0.07, and 0.79 ± 0.07, respectively. The growth rate of the control group did not significantly differ from those of the treatment groups (F ¼ 0.876, P ¼ 0.470). The proportions of unicellular algae decreased, and four- and eight-celled colonies rapidly formed in all of Daphnia watertreated populations (Fig. 2). On day 3, eight-celled colonies accounted for 28.1%, 25.0%, and 14.8% of the populations treated with test water from 1-, 3-, and 5-day-old Daphnia, respectively. Four-celled colonies were observed in the control population from day 2. Eight-celled colonies constituted 14.3% and 14.9%, 11.7% and 9.3%, and 7.1% and 7.4% of the populations treated with test water from 1-, 3-, and 5-day-old Daphnia on days 4 and 5, respectively; these results were higher than those (3.6% and 2.4% on days 4 and 5) in the control population. The morphological responses of S. obliquus were also reflected in the mean number of cells per particle. Daphnia water increased the number of cells per particle compared with the control water (Fig. 3). Furthermore, significant differences in the number of cells per particle were found among S. obliquus treated with infochemicals from Daphnia of different ages from day 3 to day 6. On day 3, the mean number of cells per particle treated with 1- and 3-day-old Daphnia-cultured water was similar (P ¼ 1.000), and an average of 4.95 ± 0.19 cells per particle was obtained; this value was significantly higher than the mean number of cells per particle treated with 5-day-old Daphnia-cultured water (4.38 ± 0.10 cells per particle, P < 0.05). On day 4, the number of cells per particle treated with 5-day-old Daphnia water was similar to that in the control population (P ¼ 0.696). A similar phenomenon was observed between the population treated with 3-day-old Daphnia cultured-water and the control population on day 5 (P ¼ 1.000). By contrast, the numbers of cells per particle treated with 1-day-old Daphniacultured water were significantly higher than those in the control water on day 4 (3.91 ± 0.21 vs. 2.80 ± 0.10, P ¼ 0.001) and day 5 (3.55 ± 0.11 vs. 2.99 ± 0.14, P ¼ 0.027). On day 6, S. obliquus treated with 1-day-old Daphnia cultured-water averaged Table 2 Body size, density, and actual feeding intensity of Daphnia magna of different ages in experiment 2. Age (day)
Body size (mm)
Density (inds L1)
Feeding intensity (106 cells day1)
2 4 6
1.002 1.534 2.110
2000 500 250
80.7 (±8.94) 75.2 (±8.53) 73.1 (±5.15)
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-1
Feeding intensity (× 106cells d )
70 a
60 50
b
40
c
30 20 10 0 1 3 5 Age of Daphnia magna (d)
Fig. 1. Actual feeding intensities of D. magna of different ages with the same biomass. Vertical lines represent 1 SE. Different lowercases indicate significant differences at P < 0.05.
Percentage of population (%)
100 80
60
40
40
20
20
0
0 0
1
2
3
4
5
6
7
8
9
3-day-old
80
100
60
40
40
20
20
0
0 1
2
unicell
3
4 5 6 7 Time (d) 2 - celled
8
0
1
2
3
4
5
6
9
7
8
9
5-day-old
80
60
0
1-day-old
80
60
100
Percentage of population (%)
100
Control
0
1
4 - celled
2
3
4 5 6 Time (d) 8 - celled
7
8
9
other
Fig. 2. Proportions of S. obliquus cells that were unicellular or two-, four-, and eight-celled colonies treated with test water derived from Daphnia of different ages with the same biomass. The other groups represent three-, five-, six-, seven-, and multi-celled colonies.
3.50 ± 0.08 cells per particle, which was significantly higher than S. obliquus treated with 5-day-old Daphnia cultured-water (2.81 ± 0.09 cells per particle, P ¼ 0.011, Fig. 3). 3.2. Experiment 2 With extended culture time, S. obliquus treated with infochemicals from Daphnia of different ages with the same feeding intensity continued to grow similarly to the control group. The growth rates of all populations ranged from 0.96 day1 to 1.02 day1; these growth rates did not significantly differ (F ¼ 0.0958, P ¼ 0.961). Unicellular algae dominated the S. obliquus population in the control group during the experiment. In all of the treatments, four- and eight-celled colonies formed rapidly on day 3; the four- and eight-celled colonies represented 31.5% and 49.4%, 16.7% and 71.4%, and 35.4% and 50.6% of the populations treated with 2-, 4- and 6-day-old Daphnia cultured-water, respectively. As culture time was extended, the proportions of four- and eight-celled colonies decreased most likely because the grazer-associated infochemicals were degraded. By contrast, the proportions remained higher than those in the control population. When data from the two-, four-, eight-celled colonies and the other colonies in a population were pooled and analyzed as a whole, the test water from Daphnia of different ages with the same feeding intensity caused similar extents of unicellular colony transformation in S. obliquus.
X. Zhu et al. / Biochemical Systematics and Ecology 61 (2015) 271e277
4.0
Day -1
4.0
b
b c
a
3.0
2.0
Day -4
4.0
b
1.0 4.0
b
Day - 5
3.5
3.5
a
a
2.0 4.0 b a
3.5
a
3.0
3.0
2.5
2.5
2.5
2.0 4.0
2.0 4.0
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Day - 3
3.0
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Day - 2
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275
a
Day -7
3.5
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Day- 6 ab
a
ab b
Control 1 3 5 Age of Daphnia magna (d)
Day -8
2.0
2.0
Control 1 3 5 Age of Daphnia magna (d)
Control 1 3 5 Age of Daphnia magna (d)
Fig. 3. Variances in the mean number of cells per particle of S. obliquus treated with the test water derived from Daphnia of different ages with the same biomass. Vertical lines represent 1 SE. Different lowercases indicate significant differences within a single graph at P < 0.05.
Consistent with the formation of colonies, the number of cells per particle of S. obliquus was significantly different between the control group and the Daphnia water-exposed populations on day 3 (F ¼ 9.679, P ¼ 0.002). When the Daphnia age data were independently analyzed, the number of cells per particle did not significantly differ; an average of 3.62 ± 0.89 was obtained among the populations treated with test water derived from Daphnia of different ages (F ¼ 0.152, P ¼ 0.861). The number of cells per particle in all of the treated S. obliquus also increased significantly compared with that in the control group on day 4 (F ¼ 41.505, P < 0.001) and day 5 (F ¼ 17.690, P < 0.001); by contrast, the treated populations did not significantly differ (F ¼ 1.082 and P ¼ 0.379 on day 4, F ¼ 1.239 and P ¼ 0.335 on day 5, Fig. 4).
5
Cells per particle
4 3
Control 2-day-old 4-day-old 6-day-old
2 1 0 1
2
3 Time (d)
4
5
Fig. 4. Variances in the mean number of cells per particle of S. obliquus treated with the test water derived from Daphnia of different ages with the same feeding intensity. Vertical lines represent 1 SE.
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4. Discussion The results confirmed that S. obliquus detected the predation risk from the infochemicals released by predators; in response, S. obliquus formed large colonies to reduce losses to grazing (Verschoor et al., 2004; Van Donk et al., 2011). The growth rates of the algal populations treated with infochemicals from Daphnia of different ages were similar to those of the untreated control populations; this result indicated that the grazer-induced colony formation in Scenedesmus is independent of growth rate. This finding is consistent with that described in previous reports, which demonstrated that infochemicals released from Daphnia do not influence the growth of S. obliquus (Lürling and Van Donk, 1997; Wu et al., 2013). These comparable growth rates of S. obliquus provided the prerequisite to compare the extent of colony formation among the different treatments. The age structures of zooplankton populations vary across developmental stages and influence the prey size preference, adaptation to environmental conditions, and interaction strengths with prey species (Sarma et al., 2005; Brose et al., 2006; Xiang et al., 2010). In experiment 1, younger Daphnia exhibited higher feeding intensities when the grazer biomass was held constant (Fig. 1). In general, body size is negatively related to metabolism of organisms (Gillooly et al., 2001; Brown et al., 2004). Young Daphnia with small body size shows a higher metabolic rate than old ones; as such, a high food threshold concentration at which respiration equals assimilation is detected (Gliwicz, 1990). In addition, more grazers were observed when the small Daphnia yielded the same biomass as the large Daphnia. These factors exacerbated the algal grazing losses to younger Daphnia at the same biomass. The water from cultures with greater algal losses caused the higher number of cells per particle in S. obliquus (Fig. 3). This observation was similar to that of Lampert et al. (1994), who demonstrated that actively feeding Daphnia induces a stronger colony formation response than starved Daphnia. Inducible algae are expected to respond morphologically by detecting chemical messengers associated with the grazing force (Lass and Spaak, 2003; Lürling, 2003a). The younger Daphnia in experiment 1 probably consumed more algae, thereby increasing the production of infochemicals; therefore, the number of cells per particle in S. obliquus increased. The results of experiment 2 confirmed this presumption. Similar amounts of infochemicals were probably produced when Daphnia of different ages consumed comparable quantities of S. obliquus. S. obliquus assessed that the grazing risk was similar and then responded with a comparable extent of colony formation by detecting these infochemicals. These results suggest that the potential grazing pressure that algae face determines the extent of grazer-induced colony formation in Scenedesmus. In aquatic systems, the predation risk may be highly variable because grazers exhibit diverse population characteristics, including population density, age structure, and spatial distribution (Kumar, 2005). With grazing loss-dependent defenses, the response of Scenedesmus to grazing pressure can be immediate and more reliable than the identification of grazer characteristics. In conclusion, S. obliquus rapidly formed colonies after exposure to infochemicals released by Daphnia of different ages with the same biomass or the same feeding intensity. With similar grazer biomass, small Daphnia exhibited higher feeding intensities than large Daphnia; thus, the former induced the formation of high proportions of eight-celled colonies and the higher number of cells per particle than the latter. 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Potential grazing intensity directly determines the extent of grazer-induced colony formation in Scenedesmus obliquus Xuexia Zhu, Haihong Nan, Qinwen Chen, Zhongqiu Wu, Xinyan Wu, Yuan Huang**, Zhou Yang* Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2015 Received in revised form 16 June 2015 Accepted 27 June 2015 Available online 6 July 2015
The genus Scenedesmus can form large colonies to provide protection against predation by herbivores. Scenedesmus has been investigated to elucidate the varying extents of colony formation in response to predation. However, limited information is available regarding the factors directly affecting the extent of colony formation responses. This study investigated the colony formation responses of Scenedesmus obliquus to infochemicals derived from Daphnia of different ages with the same biomass or feeding intensity. Results showed that water containing Daphnia infochemicals can induce the colony formation in S. obliquus regardless of age, biomass, or feeding intensity of Daphnia. The infochemicals derived from 1-day-old Daphnia caused a higher number of cells per particle than those from 5-day-old Daphnia when the grazers were of the same biomass; the infochemicals from 1-day-old Daphnia also maintained the algal colonial morph longer than those from 3-day-old Daphnia. The number of cells per particle did not differ among the algal populations treated with infochemicals from Daphnia of different ages when the feeding intensity of grazers was held constant. Therefore, potential grazing intensity directly determines the extent of the grazer-induced colony formation in S. obliquus. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Colony formation Daphnia magna Feeding intensity Grazer biomass Scenedesmus obliquus
1. Introduction In aquatic ecosystems, phytoplankton are very susceptible to herbivory by zooplankton. Many planktonic organisms sense li, 2010; Van Donk et al., 2011). potential predators by using predator-derived infochemicals (Tang, 2003; Lundgren and Grane Phytoplankton react by exhibiting morphological responses that reduce the predation risk; among these responses, colony formation is interpreted as a defense mechanism against grazing (Jakobsen and Tang, 2002; Kampe et al., 2007; Tang et al., 2008). The green alga Scenedesmus is one of the most investigated phytoplankton in terms of grazer-induced colony formation (Hessen and Van Donk, 1993; Lürling, 2003a; Yang et al., 2007). Scenedesmus responds by forming large colonies, such as an eight-celled colony, when the threat of predation by grazers is high; as a result, a collective size beyond the predation capacity of zooplankton is formed. Herbivorous zooplankton species, such as cladocerans and rotifers, trigger the unicellular colony transformation of Scenedesmus to varying extents (Lampert et al., 1994; Yang et al., 2006).
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Huang), [email protected] (Z. Yang). http://dx.doi.org/10.1016/j.bse.2015.06.035 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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In aquatic ecosystems, algae are not confronted with constant predation pressure, but rather experience rapid variation in grazing pressure. In addition to the taxonomic diversity of predators, the abundance and feeding activity of a specific grazer can vary spatially and temporally (Sterner, 1989; Viejo and Åberg, 2003). Inducible algae are expected to respond proportionally on the basis of the predation risk level because costs are associated with induced defenses (Lürling and Van Donk, 2000; Beardall et al., 2009). For instance, Lampert et al. (1994) and Lürling and Van Donk (1997) investigated the relationship between the number of Daphnia and the subsequent induced colony formation of Scenedesmus. Using Daphnia of the same age, Wu et al. (2013) found that the colony formation of Scenedesmus is dependent on the density of Daphnia; the number of cells per particle increases as the density of Daphnia increases. Zhu et al. (2015) found that large Daphnia causes the formation of high proportions of large Scenedesmus colonies when animals of different sizes were kept at the same density. High density and large body increase grazer biomass; both parameters may be responsible for the strengthened colony formation responses of Scenedesmus. In addition to grazer biomass, grazing pressure is a direct measure of the predation risks that algae face. Grazing pressure likely plays a key role in determining the extent of the grazer-induced colony formation response of Scenedesmus because the grazing activity of zooplankton triggers unicellular colony transformation (Von Elert and Franck, 1999). Lürling (2003a) found that water from cultures with high grazing rates induces the formation of more Scenedesmus obliquus colonies than water from cultures with low grazing rates. However, a high density of animals (leading to higher grazer biomass) has been used in cultures with high grazing rates, which may also contribute to the strengthened colony formation response. To eliminate such uncertainty, we conducted an experiment to evaluate the colony formation response of S. obliquus to infochemicals derived from Daphnia magna of different ages with the same biomass. We hypothesized that Daphnia at the same biomass causes similar extents of inducing colony formation; however, our results rejected this hypothesis. In a second experiment, infochemicals derived from D. magna of different ages with the same feeding intensity were used to induce unicellular colony transformation. Our results confirmed that potential grazing intensity directly determines the extent of colony formation in S. obliquus. 2. Materials and methods 2.1. Algae and zooplankton S. obliquus (FACHB-416) was obtained from the Institute of Hydrobiology, Chinese Academy of Science. The algae were precultured axenically in liquid BG-11 medium (Rippka et al., 1979) at 25 ± 1 C and illuminated at 40 mmol photons m2 s1 by using fluorescent lamps in a lightedark period of 12 h:12 h. The cladoceran D. magna, a laboratory clone maintained for more than 10 years, was cultured in beakers and fed with S. obliquus under the same conditions as those for culturing S. obliquus. 2.2. Experimental protocol Standardized bioassays were performed to determine the morphological responses of Scenedesmus to zooplankton filtrates (Hessen and Van Donk, 1993; Lürling, 2003b; Yang et al., 2007). Two experiments were conducted successively as follows: Experiment 1. To produce the test water containing infochemicals from Daphnia of different ages with the same biomass, 1-, 3- and 5-day-old D. magna were used in the experiment. The Daphnia biomass was quantified as dry weight. After three rinses with redistilled water, Daphnia at each age was dried at 80 C for 48 h, cooled at room temperature, and weighed to determine the average dry weight per age. To ensure that the Daphnia infochemicals can induce colony formation in Scenedesmus, we used Daphnia biomass of 12.8 ± 0.1 mg dry weight L1 (equal to the biomass of 5-day-old Daphnia at a density of 120 inds L1) in the experiment. Different densities of D. magna at each age (Table 1) were selected to reach the same grazer biomass. In each age, D. magna was fed on S. obliquus at a density of 1 106 cells mL1 for 24 h. The actual feeding intensity corresponding to each age of grazers was calculated on the basis of the equation of Frost (1972). To prepare the test water, we filtered the medium through a 60 mm nylon mesh and then through a 0.10 mm rinsed glass fiber filter (Millipore Corporation, USA) to remove the zooplankton and algae, respectively. A suspension culture of S. obliquus that was not exposed to Daphnia was filtered in the same way and used as control. Aliquots of exponentially growing unicellular S. obliquus from the preculture were transferred to 250 mL Erlenmeyer flasks, which were sealed with cellulose plugs. Each batch culture contained 135 mL of unicellular algal inoculum and either 15 mL of control water or 15 mL of test water. Similar initial algal concentrations were prepared for each batch culture with a
Table 1 Body size, dry weight, and density of Daphnia magna of different ages in experiment 1. Age (day)
Body size (mm)
Dry weight (mg ind1)
Density (inds L1)
1 3 5
0.788 ± 0.006 1.299 ± 0.013 2.313 ± 0.030
0.008 0.046 0.106
1590 280 120
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density of 2.0 104 cells mL1. The control group and three different treatments (containing 1-, 3-, and 5-day-old D. magnacultured water, respectively) were run in six replicates for eight days under the previously described culture conditions. The flasks were shaken thrice daily and rearranged randomly to avoid the effect of minor differences in illumination. Experiment 2. Two-, 4- and 6-day-old D. magna were used in experiment 2; the body size for each age was determined by measuring at least 30 individuals. According to the equation of Frost (1972), we obtained a series of feeding intensities corresponding to different densities of Daphnia at each age in a pre-experiment. Different numbers of Daphnia at each age were selected on the basis of these data to achieve the same feeding intensity (Table 2 for the densities and actual feeding intensities). D. magna was then fed on S. obliquus to prepare the test water, which was used to induce the colony formation of Scenedesmus in accordance with the procedures performed in experiment 1. 2.3. Algal growth and colony formation Samples (2 mL) were collected daily and preserved in Lugol's solution (2%). The abundance and the number of algal cells in different morphological particles (single cells; two-, four-, eight-celled colonies; and other colonies) were counted using a hemocytometer (Tianlong XB-K-25, China) with an optical microscope (Olympus 6V20WHAL, Japan). The growth rates were determined as the slope of the ln of cell abundance versus time from daily samples (Yang et al., 2010). The mean proportion of cells in different particles and the mean number of cells per particle were calculated from the counts. 2.4. Statistical analyses Data were presented as mean ± SE. The growth rates and feeding intensities were compared through one-way ANOVA with Daphnia ages as the factor. Differences in the number of cells per particle between groups were compared through repeated measures ANOVA. The differences obtained on each day were analyzed through multivariate analysis of variance with the Daphnia test water as the factor. Significantly different means were distinguished by a Holm-Sidak post hoc comparison test (P < 0.05). Statistical analyses were performed using SPSS 16.0 software. 3. Results 3.1. Experiment 1 During the test water preparation, the feeding intensities of 1-, 3- and 5-day-old Daphnia were 55.17 ± 5.60, 41.67 ± 1.56, and 31.01 ± 2.33 106 cells day1, respectively (Fig. 1). One-way ANOVA indicated significant differences in feeding intensities (F ¼ 11.184, P ¼ 0.009). After Daphnia-cultured water was added to the algal medium, the algal growth was comparable to that cultured in the control water. The growth rates of S. obliquus exposed to infochemicals from 1-, 3-, and 5-day-old Daphnia at the same biomass were 0.86 ± 0.11, 0.84 ± 0.07, and 0.79 ± 0.07, respectively. The growth rate of the control group did not significantly differ from those of the treatment groups (F ¼ 0.876, P ¼ 0.470). The proportions of unicellular algae decreased, and four- and eight-celled colonies rapidly formed in all of Daphnia watertreated populations (Fig. 2). On day 3, eight-celled colonies accounted for 28.1%, 25.0%, and 14.8% of the populations treated with test water from 1-, 3-, and 5-day-old Daphnia, respectively. Four-celled colonies were observed in the control population from day 2. Eight-celled colonies constituted 14.3% and 14.9%, 11.7% and 9.3%, and 7.1% and 7.4% of the populations treated with test water from 1-, 3-, and 5-day-old Daphnia on days 4 and 5, respectively; these results were higher than those (3.6% and 2.4% on days 4 and 5) in the control population. The morphological responses of S. obliquus were also reflected in the mean number of cells per particle. Daphnia water increased the number of cells per particle compared with the control water (Fig. 3). Furthermore, significant differences in the number of cells per particle were found among S. obliquus treated with infochemicals from Daphnia of different ages from day 3 to day 6. On day 3, the mean number of cells per particle treated with 1- and 3-day-old Daphnia-cultured water was similar (P ¼ 1.000), and an average of 4.95 ± 0.19 cells per particle was obtained; this value was significantly higher than the mean number of cells per particle treated with 5-day-old Daphnia-cultured water (4.38 ± 0.10 cells per particle, P < 0.05). On day 4, the number of cells per particle treated with 5-day-old Daphnia water was similar to that in the control population (P ¼ 0.696). A similar phenomenon was observed between the population treated with 3-day-old Daphnia cultured-water and the control population on day 5 (P ¼ 1.000). By contrast, the numbers of cells per particle treated with 1-day-old Daphniacultured water were significantly higher than those in the control water on day 4 (3.91 ± 0.21 vs. 2.80 ± 0.10, P ¼ 0.001) and day 5 (3.55 ± 0.11 vs. 2.99 ± 0.14, P ¼ 0.027). On day 6, S. obliquus treated with 1-day-old Daphnia cultured-water averaged Table 2 Body size, density, and actual feeding intensity of Daphnia magna of different ages in experiment 2. Age (day)
Body size (mm)
Density (inds L1)
Feeding intensity (106 cells day1)
2 4 6
1.002 1.534 2.110
2000 500 250
80.7 (±8.94) 75.2 (±8.53) 73.1 (±5.15)
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-1
Feeding intensity (× 106cells d )
70 a
60 50
b
40
c
30 20 10 0 1 3 5 Age of Daphnia magna (d)
Fig. 1. Actual feeding intensities of D. magna of different ages with the same biomass. Vertical lines represent 1 SE. Different lowercases indicate significant differences at P < 0.05.
Percentage of population (%)
100 80
60
40
40
20
20
0
0 0
1
2
3
4
5
6
7
8
9
3-day-old
80
100
60
40
40
20
20
0
0 1
2
unicell
3
4 5 6 7 Time (d) 2 - celled
8
0
1
2
3
4
5
6
9
7
8
9
5-day-old
80
60
0
1-day-old
80
60
100
Percentage of population (%)
100
Control
0
1
4 - celled
2
3
4 5 6 Time (d) 8 - celled
7
8
9
other
Fig. 2. Proportions of S. obliquus cells that were unicellular or two-, four-, and eight-celled colonies treated with test water derived from Daphnia of different ages with the same biomass. The other groups represent three-, five-, six-, seven-, and multi-celled colonies.
3.50 ± 0.08 cells per particle, which was significantly higher than S. obliquus treated with 5-day-old Daphnia cultured-water (2.81 ± 0.09 cells per particle, P ¼ 0.011, Fig. 3). 3.2. Experiment 2 With extended culture time, S. obliquus treated with infochemicals from Daphnia of different ages with the same feeding intensity continued to grow similarly to the control group. The growth rates of all populations ranged from 0.96 day1 to 1.02 day1; these growth rates did not significantly differ (F ¼ 0.0958, P ¼ 0.961). Unicellular algae dominated the S. obliquus population in the control group during the experiment. In all of the treatments, four- and eight-celled colonies formed rapidly on day 3; the four- and eight-celled colonies represented 31.5% and 49.4%, 16.7% and 71.4%, and 35.4% and 50.6% of the populations treated with 2-, 4- and 6-day-old Daphnia cultured-water, respectively. As culture time was extended, the proportions of four- and eight-celled colonies decreased most likely because the grazer-associated infochemicals were degraded. By contrast, the proportions remained higher than those in the control population. When data from the two-, four-, eight-celled colonies and the other colonies in a population were pooled and analyzed as a whole, the test water from Daphnia of different ages with the same feeding intensity caused similar extents of unicellular colony transformation in S. obliquus.
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4.0
Day -1
4.0
b
b c
a
3.0
2.0
Day -4
4.0
b
1.0 4.0
b
Day - 5
3.5
3.5
a
a
2.0 4.0 b a
3.5
a
3.0
3.0
2.5
2.5
2.5
2.0 4.0
2.0 4.0
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Day - 3
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Cells per particle
5.0
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Day - 2
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275
a
Day -7
3.5
3.5
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3.0
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Day- 6 ab
a
ab b
Control 1 3 5 Age of Daphnia magna (d)
Day -8
2.0
2.0
Control 1 3 5 Age of Daphnia magna (d)
Control 1 3 5 Age of Daphnia magna (d)
Fig. 3. Variances in the mean number of cells per particle of S. obliquus treated with the test water derived from Daphnia of different ages with the same biomass. Vertical lines represent 1 SE. Different lowercases indicate significant differences within a single graph at P < 0.05.
Consistent with the formation of colonies, the number of cells per particle of S. obliquus was significantly different between the control group and the Daphnia water-exposed populations on day 3 (F ¼ 9.679, P ¼ 0.002). When the Daphnia age data were independently analyzed, the number of cells per particle did not significantly differ; an average of 3.62 ± 0.89 was obtained among the populations treated with test water derived from Daphnia of different ages (F ¼ 0.152, P ¼ 0.861). The number of cells per particle in all of the treated S. obliquus also increased significantly compared with that in the control group on day 4 (F ¼ 41.505, P < 0.001) and day 5 (F ¼ 17.690, P < 0.001); by contrast, the treated populations did not significantly differ (F ¼ 1.082 and P ¼ 0.379 on day 4, F ¼ 1.239 and P ¼ 0.335 on day 5, Fig. 4).
5
Cells per particle
4 3
Control 2-day-old 4-day-old 6-day-old
2 1 0 1
2
3 Time (d)
4
5
Fig. 4. Variances in the mean number of cells per particle of S. obliquus treated with the test water derived from Daphnia of different ages with the same feeding intensity. Vertical lines represent 1 SE.
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4. Discussion The results confirmed that S. obliquus detected the predation risk from the infochemicals released by predators; in response, S. obliquus formed large colonies to reduce losses to grazing (Verschoor et al., 2004; Van Donk et al., 2011). The growth rates of the algal populations treated with infochemicals from Daphnia of different ages were similar to those of the untreated control populations; this result indicated that the grazer-induced colony formation in Scenedesmus is independent of growth rate. This finding is consistent with that described in previous reports, which demonstrated that infochemicals released from Daphnia do not influence the growth of S. obliquus (Lürling and Van Donk, 1997; Wu et al., 2013). These comparable growth rates of S. obliquus provided the prerequisite to compare the extent of colony formation among the different treatments. The age structures of zooplankton populations vary across developmental stages and influence the prey size preference, adaptation to environmental conditions, and interaction strengths with prey species (Sarma et al., 2005; Brose et al., 2006; Xiang et al., 2010). In experiment 1, younger Daphnia exhibited higher feeding intensities when the grazer biomass was held constant (Fig. 1). In general, body size is negatively related to metabolism of organisms (Gillooly et al., 2001; Brown et al., 2004). Young Daphnia with small body size shows a higher metabolic rate than old ones; as such, a high food threshold concentration at which respiration equals assimilation is detected (Gliwicz, 1990). In addition, more grazers were observed when the small Daphnia yielded the same biomass as the large Daphnia. These factors exacerbated the algal grazing losses to younger Daphnia at the same biomass. The water from cultures with greater algal losses caused the higher number of cells per particle in S. obliquus (Fig. 3). This observation was similar to that of Lampert et al. (1994), who demonstrated that actively feeding Daphnia induces a stronger colony formation response than starved Daphnia. Inducible algae are expected to respond morphologically by detecting chemical messengers associated with the grazing force (Lass and Spaak, 2003; Lürling, 2003a). The younger Daphnia in experiment 1 probably consumed more algae, thereby increasing the production of infochemicals; therefore, the number of cells per particle in S. obliquus increased. The results of experiment 2 confirmed this presumption. Similar amounts of infochemicals were probably produced when Daphnia of different ages consumed comparable quantities of S. obliquus. S. obliquus assessed that the grazing risk was similar and then responded with a comparable extent of colony formation by detecting these infochemicals. These results suggest that the potential grazing pressure that algae face determines the extent of grazer-induced colony formation in Scenedesmus. In aquatic systems, the predation risk may be highly variable because grazers exhibit diverse population characteristics, including population density, age structure, and spatial distribution (Kumar, 2005). With grazing loss-dependent defenses, the response of Scenedesmus to grazing pressure can be immediate and more reliable than the identification of grazer characteristics. In conclusion, S. obliquus rapidly formed colonies after exposure to infochemicals released by Daphnia of different ages with the same biomass or the same feeding intensity. With similar grazer biomass, small Daphnia exhibited higher feeding intensities than large Daphnia; thus, the former induced the formation of high proportions of eight-celled colonies and the higher number of cells per particle than the latter. By contrast, Daphnia of different ages induced the colony formation in S. obliquus with the same number of cells per particle when the feeding intensity of grazers was held constant. These results indicated that potential grazing intensity directly determines the extent of the grazer-induced colony formation in Scenedesmus. Acknowledgments This study was supported by the National Natural Science Foundation of China (31470508) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Beardall, J., Allen, D., Bragg, J., Finkel, Z.V., Flynn, K.J., Quigg, A., Rees, T.A.V., Richardson, A., Raven, J.A., 2009. Allometry and stoichiometry of unicellular, colonial and multicellular phytoplankton. New. Phytol. 181, 295e309. Brose, U., Jonsson, T., Berlow, E.L., Warren, P., Banasek-Richter, C., Bersier, L.F., Blanchard, J.L., Brey, T., Carpenter, S.R., Blandenier, M.F.C., 2006. Consumerresource body-size relationships in natural food webs. 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