Life history responses of Daphnia magna feeding on toxic Microcystis aeruginosa alone and mixed with a mixotrophic Poterioochromonas species

water research 43 (2009) 5053–5062

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Life history responses of Daphnia magna feeding on toxic Microcystis aeruginosa alone and mixed with a mixotrophic Poterioochromonas species Xue Zhang a, Trine Perlt Warming b, Hong-Ying Hu a,*, Kirsten Seestern Christoffersen b a

Environmental Simulation and Pollution Control State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China b Freshwater Biological Laboratory, University of Copenhagen, Helsingørsgade 51, 3400 Hillerød, Denmark

article info

abstract

Article history:

The toxic effects of a mixotrophic golden alga (Poterioochromonas sp. strain ZX1) and

Received 18 June 2009

a cyanobacterium Microcystis aeruginosa grazed by Poterioochromonas to a cladoceran were

Received in revised form

investigated through life history experiments using Daphnia magna. Poterioochromonas

18 August 2009

cultured in two ways (fed M. aeruginosa in an inorganic medium or grown in an organic

Accepted 19 August 2009

medium) both induced starvation-like effects on D. magna, indicating that Poter-

Available online 26 August 2009

ioochromonas is neither acutely toxic nor a good food for D. magna. Despite a microcystin-LR content of 108 mg cell1 in M. aeruginosa, no toxins were accumulated in Poterioochromonas

Keywords:

fed the cyanobacterium. The toxic effect of M. aeruginosa to D. magna was significantly

Daphnia magna

reduced in the presence of Poterioochromonas, which may be performed in two ways:

Life history response

decrease M. aeruginosa cells ingestion of D. magna by grazing on M. aeruginosa; and decrease

Microcystis aeruginosa

the toxicity of the medium by degrading the toxins released by M. aeruginosa. This study

Mixotrophy

provides new information on the interactions between a cyanobacterium and its grazer

Poterioochromonas sp.

under laboratory conditions and may increase our understanding of the ecological significance of such interactions in the aquatic food webs. ª 2009 Elsevier Ltd. All rights reserved.

1.

Introduction

Toxic cyanobacterial blooms have caused disruptions of aquatic ecosystems worldwide, as well as poisoning of livestock, natural flora and fauna communities, and even humans has been affected on many occasions (e.g., Christoffersen, 1996; Ueno et al., 1996; Jochimsen et al., 1998; Fitzgerald et al., 1999; Svrcek and Smith, 2004). Among the cyanobacterial species, Microcystis aeruginosa is one of the most common species and many strains of it produce hepatotoxic microcystins, of which almost 60 variants (e.g., MC-LR) have been identified (Park et al., 2001).

The cladoceran, Daphnia, a key species in freshwater pelagic food webs, is sensitive to microcystins and is often used in ecotoxicological tests (Lampert, 1987). Numerous studies have showed that M. aeruginosa have strong adverse effects on Daphnia, such as increased mortality, decreased growth rate, delayed maturation and decreased offspring production (e.g., Lampert, 1981; Hietala et al., 1995; Deng et al., 2008). These adverse effects may be caused in several possible ways: (1) the ‘‘food grove blocking’’ effect that markedly reduce the uptake of more suitable food by Daphnia (e.g., Nizan et al., 1986); (2) the poor nutrition value of cyanobacteria as food due to lack of essential fatty acids or lipids which

* Corresponding author. Tel.: þ86 10 6279 4005; fax: þ86 10 6279 7265. E-mail address: [email protected] (H.-Y. Hu). 0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.08.022

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decrease the growth and reproduction of Daphnia (e.g., DeMott and Mu¨ller-Navarra, 1997), and (3) the toxins produced by M. aeruginosa (e.g., MC-LR) may cause internal injuries and finally the death (e.g., DeMott and Dhawale, 1995; Rohrlack et al., 1999, 2005). Although Microcystis is generally considered difficult to graze by most zooplankton species due to the colonial form and/or toxicity, several species of protists can be active grazers. Examples are amoeba Penardochlamys sp. (Nishibe et al., 2004), flagellate Collodictyon triciliatum (Nishibe et al., 2002), Diphylleia rotans (Kim et al., 2006) and Monas-Guttula (Sugiura et al., 1992), as well as mixotrophic golden algae Poterioochromonas and Ochromonas (Cole and Wynne, 1974; Zhang et al., 1996; Ou et al., 2005; Zhang et al., 2008; Van Donk et al., 2009). Most of these studies have focused on the growth and feeding characteristic of the grazers feeding on Microcystis, while a few have dealt with the degradation of microcystins per se. One example demonstrates that Poterioochromonas can degrade the toxins produced by M. aeruginosa while consuming Microcystis cells (Zhang et al., 2008). However, as far as we know, the ecological effects caused by the grazers grazing on Microcystis have never been reported, not even in laboratory study. The results may contribute to understand the ecological role of the interactions between Microcystis and its grazers in the aquatic food webs. In our previous study (Zhang et al., 2008, 2009), a species of the mixotrophic golden algae (Poterioochromonas sp. strain ZX1), capable of grazing on M. aeruginosa and degrading MC-LR, was isolated. Since Poterioochromonas appears to be less toxic than M. aeruginosa (Kristiansen, 2005), it is possible that the toxic effects of M. aeruginosa to its grazers (e.g., Daphnia) can be reduced due to the sharp degradation of M. aeruginosa cells and the toxins under the grazing of Poterioochromonas. In this study, it was hypothesized the toxic effects of a common cyanobacterium to Daphnia may be significantly altered (reduced) after being consumed by another algae, which are also grazed by Daphnia. The purpose was to test this hypothesis during life history experiments using Daphnia magna where the toxic effects of M. aeruginosa to D. magna without and with Poterioochromonas grazing were compared. The life response of D. magna to Poterioochromonas was also examined. Furthermore, the survival and recovery of D. magna exposed to M. aeruginosa alone and with Poterioochromonas for 2 days, as well as the toxicity of the filtrates of both suspensions were examined and compared. Based on the results, the potential ecological roles of the interactions between M. aeruginosa and Poterioochromonas are discussed.

axenic and cultured as batch in 500 mL flask with lids. They were grown in a culture room with continuous light from white fluorescent lamps at 25 mmol photons m2 s1 (measured outside the culture vessels) and 20  1  C. They were harvested during exponential growth phase by centrifugation for 10 min at 3100 rpm and all the transfer was carried out under sterilized conditions. The golden alga Poterioochromonas sp. (strain ZX1) was isolated by Zhang et al. (2008) and grown under two different conditions. (1) Poterioochromonas identified as 915-ZX1 was fed M. aeruginosa cells (1–5  106 cells mL1) in BG11 medium; (2) Poterioochromonas identified as O-ZX1 was grown in a sterilized organic medium consisting of 1.0 g L1 each of glucose, tryptone and yeast extract. Both were cultured as batch in 500 mL flasks with lids and kept in the culture room as described above. Poterioochromonas 915-ZX1 fed M. aeruginosa was harvested by centrifugation for 10 min at 3100 rpm after M. aeruginosa cells were grazed down (M. aeruginosa density decreased to lower than the detection limit of 104 cells mL1) typically after 4 days. Poterioochromonas O-ZX1 grown in the organic medium was also harvested by centrifugation for 10 min at 3100 rpm when it reached the log phase after 4 days. All algal cells (M. aeruginosa, S. acutus, 915-ZX1 and O-ZX1) harvested by centrifugation were resuspended in the synthetic zooplankton medium ADaM (Klu¨ttgen et al., 1994) before being fed to the Daphnia cultures. The cladoceran species D. magna Straus was originally isolated from Langedam Denmark in 1978, and was kindly supplied by O. Kusk (Technical University of Denmark, Lyngby, Copenhagen). The culture has been maintained and used in the experiments at the Freshwater Biological Laboratory for many years (Warming et al., 2009). The D. magna clone was cultured in ADaM medium with S. acutus as the sole food source. Prior to the experiments 20 neonates were harvested from well-fed stock cultures, and transferred into 750 mL glass jars containing 500 mL suspension of 2 mg C L1 S. acutus. The cultures were kept in a temperature controlled room at 20  1  C, under dim light conditions. The food suspension was renewed daily and any offspring were removed from the stock culture. The animals were maintained under these conditions for at least 2 weeks and served as mothers for the animals used in the experiments. All neonates for a given experiment were taken from a single brood and within 24 h after birth.

2.2.

2.

Materials & methods

2.1.

Cultures

M. aeruginosa FACHB915 was purchased from the Freshwater Algae Culture collection of Institute of Hydrobiology (FACHB) (Wuhan, Hubei Province, China) and was cultured in sterilized BG11 medium (Rippka et al., 1979). The green alga Scenedesmus acutus Meyen (originally obtained from the Max Planck Institute for Limnology, Plo¨n, Germany) was cultured in sterilized Z8 medium (Kotai, 1972). Both M. aeruginosa and S. acutus were

Quantification of algal cells

Subsamples (200 mL per sample) of the M. aeruginosa and Poterioochromonas cultures were fixed with buffered glutaraldehyde (1% final concentration) and the cell densities were determined using a hemocytometer using light microscopy (OLYMPUS, BH-2, magnification: 400). Dry weights of M. aeruginosa and Poterioochromonas were measured as 1  108 mg cell1 and 8  108 mg cell1, respectively. Carbon content was calculated as 50% of the dry mass (Geller, 1975). The concentrations of S. acutus were estimated according to previously established relations between algal biovolume and light extinction (800 nm) as outlined in Rohrlack et al. (2001).

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2.3.

Measurement of MC-LR concentrations

The concentrations of MC-LR in the filtrates of M. aeruginosa suspensions with and without Poterioochromonas were analyzed using a commercial ELISA technique (EnSys, UK) based on polyclonal antibodies raised against microcystin (Chu et al., 1989) as described by Thostrup and Christoffersen (1999). Subsamples (5 mL per sample) were taken after D. magna in the suspension was dead and stored in 20  C refrigerator. Briefly, raw or diluted (dilution ratio of 50) samples of 100 mL were added to test tubes pre-coated with polyclonal antibodies followed by addition of an enzymelinked substrate. The antibodies were specific to microcystinLR (MC-LR) at low concentrations. Three replicates were tested for each sample.

2.4.

Table 2 – Median survival time of D. magna fed with eight different algal suspensions and in one starvation control. Algal suspension

Food concentration (mg C L1)

Median survival time (d)  SE (N ¼ 10)

Starvation control S. acutus M. aeruginosa M. aeruginosa M. aeruginosa M. aeruginosa þ Poterioochromonas M. aeruginosa þ Poterioochromonas M. aeruginosa þ Poterioochromonas Poterioochromonas

0 2 1 5 10 1 þ 0.8

5.5  0.5 >21 2.3  0.2 2.5  0.2 2.5  0.2 4.4  0.3

5 þ 0.8

3.6  0.4

10 þ 0.8

3.8  0.3

0.8

5.4  0.4

Life history experiment

Two sets of experiments were carried out: a) Life history responses of D. magna to different algal cultures. This was tested using four different algal food suspensions (S. acutus (i.e., high quality food conditions), Poterioochromonas 915-ZX1, Poterioochromonas O-ZX1 and M. aeruginosa) of the same food concentration of 5 mg C L1 and a starvation control (no food). The food suspensions are outlined in Table 1. b) Life history response of D. magna to M. aeruginosa alone and mixed with Poterioochromonas. This was tested using eight different algal suspensions and a starvation control as outlined in Table 2. The suspension of S. acutus (2 mg C L1) in the control group was renewed daily for 21 days while suspensions in other groups were not renewed. Also, the densities of M. aeruginosa and Poterioochromonas were monitored daily by randomly selecting three bottles in the seven groups with M. aeruginosa or Poterioochromonas or both. All experiments were carried out in 25 mL glass vials, containing 20 mL algal suspension in ADaM medium or pure ADaM medium (starvation control). Neonates were sampled randomly from the stock cultures and transferred individually into glass vials using a plastic pipette. Ten replicates were prepared for each group. The experimental cultures were maintained under similar conditions as the stock cultures of

Table 1 – Median survival time of D. magna fed with four different algal cultures and in one starvation control. Algal suspension Food concentration Median survival time (d)  SE (N ¼ 10) (mg C L1) Starvation control S. acutus Poterioochromonas 915-ZX1 Poterioochromonas O-ZX1 M. aeruginosa

0 5 5

4.7  0.4 >21 5.1  0.5

5

5.5  0.4

5

2.1  0.3

D. magna (i.e., at 20  1  C and dim light), but placed on a shaking table (50 rpm) to keep the algae in suspension. The algal suspensions were renewed daily unless otherwise stated as in b (see above). The production of offspring and mortality were monitored daily for 21 days. A daphnid was considered dead if no movements were observed during 30 s of intensive disturbance (Rohrlack et al., 2001). The body length defined as the distance from the centre of the eye to the base of the tail spine of all daphnids was measured using a stereo microscope (Kyowa SD/2PL) every three days. The pH values and the concentrations of dissolved oxygen in suspensions before and after one day of culture were measured with pH meter (PHM85, PRECISION) and an oxygen sensor (PA2000, Unisense ˚ rhus Denmark). The concentrations of Picoammeter A/S, A dissolved oxygen in all algal suspensions were around 9 mg L1 (close to 100% saturation) before and after one day of culture and pH ranged within 6.9–8.2.

2.5.

Acute toxicity experiment with algal filtrate

O-ZX1 and M. aeruginosa were centrifuged and resuspended in ADaM medium to final densities of 106 cells mL1 (i.e., 40 mg C L1) for Poterioochromonas O-ZX1 and 107 cells mL1 (i.e., 50 mg C L1) for M. aeruginosa. After a week of incubation, the suspensions of Poterioochromonas O-ZX1 and M. aeruginosa in ADaM medium, as well as Poterioochromonas O-ZX1 in organic medium (40 mg C L1), were centrifuged at 3100 rpm for 10 min. The supernatant was then filtered (0.2 mm) before the acute toxicity test. Four groups were prepared and included: controls (ADaM medium), filtrates of Poterioochromonas O-ZX1 grown in ADaM medium, M. aeruginosa grown in ADaM medium and Poterioochromonas O-ZX1 grown in organic medium. These experiments were carried out in 25 mL glass vials, containing 20 mL filtrate of algal suspensions or pure ADaM medium (control). Five newly released neonates were sampled randomly from the stock cultures and transferred into a glass vial. Four replicates were prepared for each group. The experimental cultures were maintained under the same conditions as described for the life history experiments above. The mortality was monitored daily for 2 days.

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Experiment time (days)

Pretreated 2 d

M. aeruginosa 2 mg C L-1, 30 Daphnia

Experiment start

0

Pretreated Daphnia

10 ADaM, 10 Daphnia S. acutus 2mg C L-1 , 10 Daphnia

Algal suspension filtrate, 10 Daphnia

M. aeruginosa 2 mg C L-1 added 1 mg C L -1

Pretreated Daphnia

Poterioochromonas, 30 Daphnia

ADaM, 10 Daphnia S. acutus 2mg C L-1, 10 Daphnia

Algal suspension filtrate, 10 Daphnia Algal suspension, 10 Daphnia

Fig. 1 – Experimental design (see Methods for details).

2.6. Toxin accumulated in D. magna and toxicity of filtrates This experiment was designed to compare the toxins accumulated in D. magna exposed to M. aeruginosa with and without Poterioochromonas, and the toxicity of the filtrates of these algal suspensions (Fig. 1). Firstly, three neonates were transferred to one 25 mL glass vial with 20 mL suspension of only M. aeruginosa (2 mg C L1) or M. aeruginosa (2 mg C L1) with Poterioochromonas (1 mg C L1). Ten replicates were prepared for each suspension and all were maintained under the conditions described above for 2 days. Meanwhile, a starvation control group of one neonate in a glass vial with 10 replicates was prepared. After 2 days, the live D. magna in the two suspensions of M. aeruginosa (single and mixed) were transferred to ADaM medium (starvation condition) or suspension of S. acutus (2 mg C L1) with 10 D. magna in each group cultured as the life history experiments (i.e., one D. magna in a glass vial, same culture condition and medium renewed daily). The mortality and growth of these D. magna were observed daily. The survival of these D. magna may represent injury or cell damage caused by the toxins accumulated from the 2-day exposure to M. aeruginosa. After removing all live D. magna from the two M. aeruginosa (single, mixed) suspensions, the toxicity of these suspension filtrates was test. The suspensions were centrifuged at 3100 rpm for 10 min and the supernatant was then filtered (0.2 mm). The toxicity of these two filtrates, as well as the suspension of M. aeruginosa with Poterioochromonas, was tested in 25 mL glass vials, containing 20 mL filtrate or algal suspensions, with 2 neonates in one glass vial and five replicates for each sample. The experimental cultures were maintained under the same conditions as described for the life history experiments above. The mortality was monitored daily for 10 days. These results should reflect effects of the toxins released to the medium.

2.7.

Data analysis

The median survival time of Daphnia was calculated in all life history experiments by the probit analysis method using Biostat (AnalystSoft, version 2008). The statistical significance of possible differences between survival functions for two treatments was determined with log-rank tests, using GraphPad Prism 5.01 (GraphPad software Inc.) and Student’s t-test by comparing the median survival time.

3.

Results

3.1. Life history responses of D. magna to different algal cultures Significant differences in life history responses were observed for D. magna fed four different algae cultures (S. acutus, Poterioochromonas 915-ZX1, Poterioochromonas O-ZX1 and M. aeruginosa) as well as a starvation control (Fig. 2). D. magna fed S. acutus grew well (the length increased up to 3.8 mm on the 21st day) and all animals survived throughout the experimental period (21 days) and produced numerous offspring (94  5.7 juveniles per D. magna). This is in contrast to the other food treatments where D. magna all died within 10 days and no growth and no offspring were observed during the whole period. The median survival time of D. magna was calculated and likewise showed marked differences (Table 1). For D. magna fed M. aeruginosa culture, the median survival time of 2.1 days was significantly shorter than that in the starvation control (4.7 days) ( p < 0.05, t-test), implying that M. aeruginosa was acutely toxic to D. magna. In the suspensions of Poterioochromonas cultured in two ways, no significant differences in D. magna survival were observed between the two groups ( p > 0.05, t-test). Also, the median survival days of D. magna in both groups fed the flagellate were similar with

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Fig. 2 – Survival (percent of the initial number) of D. magna fed four different algal cultures and in one starvation control. Each data point represents 10 replicates.

that in the starvation control ( p > 0.05, t-test), but significantly shorter than that in the normal food control group with S. acutus ( p < 0.05, t-test), indicating that Poterioochromonas is not acutely toxic to D. magna, but that it may be inadequate nutritional or inedible for D. magna.

3.2. Life history response of D. magna to M. aeruginosa alone and mixed with Poterioochromonas The life history responses of D. magna fed different concentrations of M. aeruginosa were tested under two conditions: without and with Poterioochromonas. All D. magna fed S. acutus survived throughout the incubation period (21 days), but in the

other groups all animals died within 8 days (Fig. 3). The median survival time differed substantially between the different algal food suspensions (Table 2). In the three M. aeruginosa suspensions of 1, 5 and 10 mg C L1, D. magna all died quickly (within 2–3 days) and no significant differences among the three groups were observed ( p > 0.05, t-test). The median survival time of D. magna fed M. aeruginosa (2.3–2.5 days) was significantly lower than that of the starvation control group (5.5 days) ( p < 0.05, t-test). In the three suspensions with M. aeruginosa (1, 5 and 10 mg C L1) and Poterioochromonas, the median survival time of D. magna (3.6–4.4 days) was significantly longer than those in the suspensions with only M. aeruginosa ( p < 0.05, t-test). Also, the

Fig. 3 – Survival (percent of the initial number) of D. magna fed eight different algal suspensions (single and mixed) and in one starvation control (MA represents M. aeruginosa, Po represents Poterioochromonas). Each data point represents 10 replicates.

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first few days and then dropped to the initial levels, with average densities of 2  104, 6  104, 9  104 cells mL1 (equivalent to 0.8, 2.4, 3.6 mg C L1), respectively.

3.3.

Acute toxicity of the algal culture filtrates

No D. magna died in the four tested filtrates i.e., the control filtrate (ADaM medium), the filtrate from Poterioochromonas O-ZX1 grown in ADaM medium, the filtrate from M. aeruginosa grown in ADaM medium and, finally, the filtrate from Poterioochromonas O-ZX1 grown in an organic medium for 2 days.

3.4. Toxin accumulated in D. magna and toxicity of filtrates

Fig. 4 – Density change of M. aeruginosa and Poterioochromonas during the incubation (MA represents M. aeruginosa, Po represents Poterioochromonas). Values are means ± standard errors (N [ 3).

median survival time of D. magna fed a lower concentration of M. aeruginosa (1 mg C L1) was significant longer than those in the other two groups ( p < 0.05, t-test). In the suspension of Poterioochromonas, the median survival time of D. magna was 5.4 days, which was similar to that of the starvation control ( p > 0.05, t-test), but significantly longer than those in suspensions with M. aeruginosa ( p < 0.05, t-test). The cell densities of M. aeruginosa and Poterioochromonas were monitored daily and it appeared that M. aeruginosa kept the initial levels throughout the incubation period in the three suspensions with only M. aeruginosa (Fig. 4). However, in the other three suspensions with M. aeruginosa and Poterioochromonas, M. aeruginosa decreased quickly due to Poterioochromonas grazing and the densities dropped to below 104 cells mL1 (the detection limit) after 1, 5 and 6 days, respectively. Meanwhile, Poterioochromonas densities increased in the

D. magna exposed to suspensions of M. aeruginosa alone (DM ) and mixed with Poterioochromonas (DM&P) for 2 days had different survival responses when transferred to ADaM medium or suspensions of S. acutus (Fig. 5). D. magna fed M. aeruginosa alone (DM ) for 2 days all died quickly in both ADaM medium and S. acutus suspensions and the life spans in both groups were significantly shorter than those in the starvation control ( p < 0.05, log-rank test). Only one DM in S. acutus suspension survived for 6 days, but no growth was observed during the whole period. D. magna fed M. aeruginosa mixed with Poterioochromonas (DM&P) for 2 days all survived significantly longer than those DM in both ADaM medium and S. acutus suspensions ( p < 0.05, log-rank test), but the life spans of DM&P transferred to ADaM medium (i.e., starvation) were significantly shorter than those in the starvation control ( p < 0.05, log-rank test). In contrast, 80% of DM&P recovered in S. acutus and body length increased from 1 mm on the 1st day to 2.4 mm on the 10th day (Fig. 6). All the living DM&P carried eggs and three of them produced 6–9 juveniles on the 10th day. The filtrates of M. aeruginosa suspensions with and without Poterioochromonas after culturing D. magna for 2 days differed in toxicity (Fig. 7). All D. magna died on the 2nd day in the filtrates of M. aeruginosa suspension, significantly faster than those in starvation control ( p < 0.05, log-rank test) and also, quite different from the filtrate of M. aeruginosa suspended in ADaM medium in the acute toxicity experiments, which may be caused by the facts that more toxins were released under the grazing of Daphnia (e.g., Jang et al., 2003, 2008). In contrast, D. magna in the filtrates of M. aeruginosa with Poterioochromonas survived as long as those in the starvation control ( p > 0.05, log-rank test), while D. magna in the suspensions of M. aeruginosa with Poterioochromonas survived longer than those in the starvation control ( p < 0.05, log-rank test). The MC-LR concentrations of the filtrates of M. aeruginosa suspensions (56.9  0.9 mg L1) were significantly higher than those in the filtrates of M. aeruginosa mixed with Poterioochromonas suspensions (6.0  0.5 mg L1) ( p < 0.05, t-test).

4.

Discussion

The toxicities of cultured M. aeruginosa and Poterioochromonas were tested using classic life history experiments with D. magna under the assumption that food particles must be

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Fig. 5 – Survival (percent of the initial number) of D. magna exposed to M. aeruginosa alone (DM ) and mixed with Poterioochromonas (DM&P) for 2 days after being transferred into ADaM medium (Starvation) and S. acutus suspension. Starvation control represents the survival of D. magna in ADaM medium without being exposed to M. aeruginosa.

considered toxic when the consumer feeding on them has a shorter life span than starving animals (Lampert, 1981). The survival time of D. magna fed M. aeruginosa alone was significantly shorter than that found for the starvation controls (no food), implying that M. aeruginosa was acutely toxic to D. magna. This is consistent with numerous of former studies (e.g., DeMott and Dhawale, 1995; Hietala et al., 1997; Rohrlack et al., 1999, 2001). D. magna fed Poterioochromonas sp. ZX1 survived longer than or at least as long as those in starvation control (depending on algae concentration), but significantly longer than those fed M. aeruginosa. It is likewise reasonable to conclude that Poterioochromonas is not acutely toxic to

D. magna. However, Poterioochromonas is not a very good food source for D. magna compared with S. acutus. There were no significant differences in the life responses of D. magna to Poterioochromonas fed M. aeruginosa (915-ZX1) and grown in an organic medium (O-ZX1), suggesting that no toxins (e.g., MC-LR) were accumulated in Poterioochromonas 915-ZX1 from M. aeruginosa. This result is consistent with the previous findings that no MC-LR was accumulated in the cells of Poterioochromonas when grazing on M. aeruginosa (Zhang et al., 2008). Meanwhile, other substances in M. aeruginosa other than microcystins may be poisonous to Daphnia (Rohrlack et al., 1999; Kaebernick et al., 2001; Lu¨rling, 2003), and such

Fig. 6 – Body length of D. magna exposed to M. aeruginosa alone (DM ) and mixed with Poterioochromonas (DM&P) for 2 days after being transferred into S. acutus suspension. Values are means ± SE of 8–10 replicates for DM&P while only one replicate survived for DM.

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Fig. 7 – Survival (percent of initial) of D. magna neonates in starvation control (no food), filtrate of M. aeruginosa suspension (Filtrate of MA), filtrate of M. aeruginosa with Poterioochromonas suspension (Filtrate of MA&Po) and suspension of M. aeruginosa with Poterioochromonas after culturing D. magna for 2 days.

substances are not accumulated in Poterioochromonas, either. Thus, there seem to be no bio-transfer of toxins from M. aeruginosa to D. magna through Poterioochromonas. Interesting results on the toxicity of mixotrophic Poterioochromonas malhamensis to Daphnia ambigua were reported by Leeper and Porter (1995). They found that P. malhamensis (104 and 105 cells mL1, equivalent to 0.4 and 4 mg C L1) cultured autotrophically in an inorganic medium provided sufficient nutrition for survival and reproduction of D. ambigua. However, the same strain of P. malhamensis turned out to be toxic and killed all D. ambigua in five days when it was grown heterotrophically on an organic medium. In contrast, Boenigk and Stadler (2004) found that two strains of P. malhamensis fed heat-killed bacteria (Gammaproteobacterium Listonella pelagia CB5) showed little toxicity to D. magna. The median survival days of D. magna fed the two strains of P. malhamensis (1.5  104 cells mL1, equivalent to 0.6 mg C L1) were 17 days and 12 days, respectively, which were significantly longer than that in the starvation control (5 days). These results may not be directly comparable with our findings as large Daphnia were used in their studies, while neonates that were less than 24 h old were applied in this study. Also, different strains of Daphnia and Poterioochromonas, as well as different experimental conditions were used in these studies. However, the responses of Daphnia in Boenigk and Stadler (2004) and this study are similar, which gives further support to the idea that Poterioochromonas is not acutely toxic to but also not a good food source for D. magna. The toxicity of M. aeruginosa to D. magna was changed significantly in the presence of Poterioochromonas, because M. aeruginosa were quickly grazed by Poterioochromonas. The life span of D. magna was prolonged when a toxic food item is consumed by another food item. Despite this, D. magna still died earlier in mixed food suspensions compared with cases fed only Poterioochromonas as food or no food at all. This may be caused by small amounts of toxins from the M. aeruginosa

cells ingested by D. magna during the first few days, or to toxins released by M. aeruginosa to the medium. D. magna fed M. aeruginosa alone for 2 days all died significantly faster in either ADaM medium or S. acutus suspensions than those in the starvation control, with 80–90% dead on the first day after being transferred to new medium, implying that these D. magna got cell damages by the toxins ingested with M. aeruginosa cells. These results are in accordance with previous studies, which showed that Daphnia parvula accumulated microcystins in their bodies when grazing on toxic Microcystis (Mohamed, 2001) and microcystins ingested with Microcystis cells were toxic to Daphnia (Rohrlack et al., 2001). Only one of these D. magna survived in S. acutus suspension for 6 days, but no growth was observed. Based on previous study that the alimentary canal of D. magna may be the target organ affected by exposure of microcystins (Chen et al., 2005), it is likely that the surviving D. magna had suffered damage to the alimentary canal and could not digest S. acutus, which resulted in starvation and no growth. In contrast, D. magna fed M. aeruginosa mixed with Poterioochromonas (DM&P) for 2 days all survived significantly longer than those fed M. aeruginosa alone (DM ) after being transferred to ADaM medium or S. acutus suspensions, especially for those transferred into S. acutus suspensions. However, both DM&P and DM died significantly faster in starvation than those in the starvation control. These results implied that fewer toxins were ingested by DM&P compared with DM and also, the damage to D. magna caused by the fewer toxins could be recovered when fed enough good-quality food (S. acutus). The filtrates of M. aeruginosa suspensions were toxic and killed all neonates in 2 days, while the filtrates and suspensions of M. aeruginosa with Poterioochromonas were not toxic since D. magna survived in both filtrates as long as in the starvation control. These results were consistent with the facts that more toxins (56.9 mg L1 MC-LR) were released to the medium when D. magna feeding on M. aeruginosa, while much less toxins (6.0 mg L1 MC-LR) were in the

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filtrates of the mixed suspensions of M. aeruginosa and Poterioochromonas. The toxic effects decreased in both filtrates and suspensions in the presence of Poterioochromonas may be contributed to the degradation of the toxins by Poterioochromonas, which had been reported in our previous study (Zhang et al., 2008). However, the concentrations of MC-LR in both filtrates were much lower than those found toxic to Daphnia in previous studies, i.e., the 48-h LC50 of MC-LR to three Daphnia ranged from 9.6 to 21.4 mg L1 in acute toxicity experiments (Demott et al., 1991); no clear response of Daphnia pulicaria was observed when exposed to microcystins of 50–500 mg L1 (Ghadouani et al., 2004). It is possible to deduce that MC-LR in such low concentrations could not cause mortality of D. magna in this filtrate experiment and there must be something else more toxic to D. magna, which has been reported by many researchers before (i.e., Jungmann, 1992; Rohrlack et al., 1999; Kaebernick et al., 2001; Lu¨rling, 2003). Furthermore, these compounds toxic to D. magna must be in lower concentration in the filtrates of the mixed suspension than that in the filtration of M. aeruginosa suspension. These could be caused indirectly by the reduction in density of the M. aeruginosa, resulting in a lower amount of cyanobacterial cells that may release such compounds or/and due to the degradation of these compounds by Poterioochromonas. Based on these results, it is possible to conclude that Poterioochromonas may decrease the toxic effects of M. aeruginosa to D. magna in two ways: (1) decrease M. aeruginosa cells ingestion of D. magna by grazing on M. aeruginosa; (2) decrease the toxicity of the medium by degrading the toxins released by M. aeruginosa. M. aeruginosa has the capacity to proliferate and being a dominant component in the pelagic food web in many freshwater ecosystems where it may have a negative influence on the success of zooplankton populations ability to proliferate which eventually can alter the zooplankton community structure (e.g., Fulton and Paerl, 1988; Sartonov, 1995; Smith and Gilbert, 1995; Christoffersen, 1996). However, few attempts have been made to elucidate the influences of organisms that actively graze on M. aeruginosa. In this study, it is for the first reported that the toxic effect of M. aeruginosa to D. magna is reduced under the grazing of Poterioochromonas. The results may help to understand the complicated interactions between M. aeruginosa and its grazers in the aquatic food webs.

5.

Conclusions

(1) M. aeruginosa was acutely toxic to D. magna, since D. magna fed M. aeruginosa survived significantly shorter than those in the starvation control. Poterioochromonas was neither acutely toxic nor a good food for D. magna, as Poterioochromonas induced starvation-like effects on D. magna. Despite a microcystin-LR content of 108 mg cell1 in M. aeruginosa, no toxins were accumulated in Poterioochromonas from the cyanobacterium. (2) M. aeruginosa mixed with Poterioochromonas was grazed down quickly, while it kept in the initial density levels in the single culture. D. magna survived significantly longer in the mixed cultures of M. aeruginosa and Poterioochromonas

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than those fed single M. aeruginosa. Poterioochromonas may decrease the toxic effects of M. aeruginosa to D. magna in two ways: decrease M. aeruginosa cells ingestion of D. magna by grazing on M. aeruginosa; and decrease the toxicity of the medium by degrading the toxins released by M. aeruginosa.

Acknowledgements This study was funded by China National Science Fund for Distinguished Young Scholars (No.50825801) and NSFC-JST joint-project (No.50721140017). Additionally, we thank the China scholarship Council, as well as the Freshwater Biological Laboratory, University of Copenhagen, for financial support.

references

Boenigk, J., Stadler, P., 2004. Potential toxicity of chrysophytes affiliated with Poterioochromonas and related Spumella-like flagellates. Journal of Plankton Research 26 (12), 1507–1514. Chen, W., Song, L.R., Ou, D.Y., Gan, N.Q., 2005. Chronic toxicity and responses of several important enzymes in Daphnia magna on exposure to sublethal microcystin-LR. Environmental Toxicology 20 (3), 323–330. Christoffersen, K., 1996. Ecological implications of cyanobacterial toxins in aquatic food webs. Phycologia 35 (6 Suppl.), 42–50. Chu, F.S., Huang, X., Wei, R.D., Carmichael, W.W., 1989. Production and characterization of antibodies against microcystins. Applied and Environmental Microbiology 55 (8), 1928–1933. Cole, G.T., Wynne, M.J., 1974. Endocytosis of Microcystis aeruginosa by Ochromonas danica. Journal of Phycology 10 (4), 397–410. DeMott, W.R., Dhawale, S., 1995. Inhibition of in vitro protein phosphatase activity in three zooplankton species by microcystin-LR, a toxin from cyanobacteria. Archiv Fu¨r Hydrobiologie 134 (4), 417–424. DeMott, W.R., Mu¨ller-Navarra, D.C., 1997. The importance of highly unsaturated fatty acids in zooplankton nutrition: evidence from experiments with Daphnia, a cyanobacterium and lipid emulsions. Freshwater Biology 38 (3), 649–664. Demott, W.R., Zhang, Q.X., Carmichael, W.W., 1991. Effects of toxic cyanobacteria and purified toxins on the survival and feeding of a copepod and 3 species of Daphnia. Limnology and Oceanography 36 (7), 1346–1357. Deng, D.G., Xie, P., Zhou, Q., Yang, H., Guo, L.G., Geng, H., 2008. Field and experimental studies on the combined impacts of cyanobacterial blooms and small algae on crustacean zooplankton in a large, eutrophic, subtropical, Chinese lake. Limnology 9 (1), 1–11. Fitzgerald, D.J., Cunliffe, D.A., Burch, M.D., 1999. Development of health alerts for cyanobacteria and related toxins in drinking water in South Australia. Environmental Toxicology 14 (1), 203–209. Fulton, R.S., Paerl, H.W., 1988. Effects of the blue-green alga Microcystis aeruginosa on zooplankton competitive relations. Oecologia 76 (3), 383–389. Geller, W., 1975. Die Nahrungsaufnahme von Daphnia pulex in Abha¨ngigkeit von der Futterkonzentration, der Temperatur, der Ko¨rpergro¨ße und dem Hungerzustand der Tiere. Archiv fu¨r Hydrobiologie Suppl. 48, 47–107. Ghadouani, A., Pinel-Alloul, B., Plath, K., Codd, G.A., Lampert, W., 2004. Effects of Microcystis aeruginosa and purified

5062

water research 43 (2009) 5053–5062

microcystin-LR on the feeding behavior of Daphnia pulicaria. Limnology and Oceanography 49 (3), 666–679. Hietala, J., Laure´n-Ma¨a¨tta¨, C., Walls, M., 1997. Life history responses of Daphnia clones to toxic Microcystis at different food levels. Journal of Plankton Research 19 (7), 917–926. Hietala, J., Reinikainen, M., Walls, M., 1995. Variation in life history responses of Daphnia to toxic Microcystis aeruginosa. Journal of Plankton Research 17 (12), 2307–2318. Jang, M.H., Ha, K., Joo, G.J., Takamura, N., 2003. Toxin production of cyanobacteria is increased by exposure to zooplankton. Freshwater Biology 48 (9), 1540–1550. Jang, M.H., Ha, K., Takamura, N., 2008. Microcystin production by Microcystis aeruginosa exposed to different stages of herbivorous zooplankton. Toxicon 51 (5), 882–889. Jochimsen, E.M., Carmichael, W.W., An, J., Cardo, D.M., Cookson, S.T., Holmes, C.E.M., Antunes, M.B., Filho, D.A.M., Lyra, T.M., Barreto, V.S.T., Azevedo, S.M.F.O., Jarvis, W.R., 1998. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. New England Journal of Medicine 338 (13), 873–878. Jungmann, D., 1992. Toxic compounds isolated from Microcystis pcc7806 that are more active against Daphnia than 2 Microcystins. Limnology and Oceanography 37 (8), 1777–1783. Kaebernick, M., Rohrlack, T., Christoffersen, K., Neilan, B.A., 2001. A spontaneous mutant of microcystin biosynthesis: genetic characterization and effect on Daphnia. Environmental Microbiology 3 (11), 669–679. Kim, B.R., Nakan, S., Kim, B.H., Han, M.S., 2006. Grazing and growth of the heterotrophic flagellate Diphylleia rotans on the cyanobacterium Microcystis aeruginosa. Aquatic Microbial Ecology 45 (2), 163–170. Klu¨ttgen, B., Dulmer, U., Engels, M., Ratte, H.T., 1994. An artificial freshwater for the culture of zooplankton. Water Research 28 (3), 743–746. Kotai, J., 1972. Instructions for Preparation of Modified Nutrient Solution Z8 for Algae. Publication B-11/69 1-5. Norwegian Institute for Water Research, Bergen, Norway. Kristiansen, J., 2005. Golden Algae, a Biology of Chrysophytes. pp. 1–49. A.R.G. Gantner Verlag, Rugell Lichtenstein. Lampert, W., 1981. Inhibitory and toxic effects of blue-green algae on Daphnia. Internationale Revue der gesamten hydrobiologie 66 (3), 285–298. Lampert, W., 1987. Feeding and nutrition in Daphnia. In: Peters, R.H., De Bernardi, R. (Eds.), Daphnia. Mem. Instituto Italiano di Idrobiologia, Italy, pp. 143–192. Leeper, D.A., Porter, K.G., 1995. Toxicity of the mixotrophic chrysophyte Poterioochromonas malhamensis to the cladoceran Daphnia ambigua. Archiv Fu¨r Hydrobiologie 134 (2), 207–222. Lu¨rling, M., 2003. Daphnia growth on microcystin-producing and microcystin-free Microcystis aeruginosa in different mixtures with the green alga Scenedesmus obliquus. Limnology and Oceanography 48 (6), 2214–2220. Mohamed, Z.A., 2001. Accumulation of cyanobacterial hepatotoxins by Daphnia in some Egyptian irrigation canals. Ecotoxicology and Environmental Safety 50 (1), 4–8. Nishibe, Y., Kawabata, Z., Nakano, S., 2002. Grazing on Microcystis aeruginosa by the heterotrophic flagellate Collodictyon triciliatum in a hypertrophic pond. Aquatic Microbial Ecology 29 (2), 173–179. Nishibe, Y., Manage, P.M., Kawabata, Z., Nakano, S., 2004. Trophic coupling of a testate amoeba and Microcystis species in a hypertrophic pond. Limnology 5 (2), 71–76. Nizan, S., Dimentman, C., Shilo, M., 1986. Acute toxic effects of the cyanobacterium Microcystis aeruginosa on Daphnia magna. Limnology and Oceanography 31 (3), 497–502.

Ou, D.Y., Song, L.R., Gan, N.Q., Chen, W., 2005. Effects of microcystins on and toxin degradation by Poterioochromonas sp. Environmental Toxicology 20 (3), 373–380. Park, H.D., Sasaki, Y., Maruyama, T., Yanagisawa, E., Hiraishi, A., Kato, K., 2001. Degradation of the cyanobacterial hepatotoxin microcystin by a new bacterium isolated from a hypertrophic lake. Environmental Toxicology 16 (4), 337–343. Rippka, R., Deruelles, J., Waterbury, J.B., 1979. Generic assignment, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111, 1–61. Rohrlack, T., Christoffersen, K., Dittmann, E., Nogueira, I., Vasconcelos, V., Borner, T., 2005. Ingestion of microcystins by Daphnia: intestinal uptake and toxic effects. Limnology and Oceanography 50 (2), 440–448. Rohrlack, T., Dittmann, E., Borner, T., Christoffersen, K., 2001. Effects of cell-bound microcystins on survival and feeding of Daphnia spp. Applied and Environmental Microbiology 67 (8), 3523–3529. Rohrlack, T., Dittmann, E., Henning, M., Borner, T., Kohl, J.G., 1999. Role of microcystins in poisoning and food ingestion inhibition of Daphnia galeata caused by the cyanobacterium Microcystis aeruginosa. Applied and Environmental Microbiology 65 (2), 737–739. Sartonov, A., 1995. Effects of Microcystis aeruginosa on interference competition between Daphnia pulex and Keratella cochlearis. Hydrobiologia 307 (1–3), 117–126. Smith, A.D., Gilbert, J.J., 1995. Relative susceptibilities of rotifers and cladocerans to Microcystis aeruginosa. Archiv Fu¨r Hydrobiologie 132 (3), 309–336. Sugiura, N., Inamori, Y., Ouchiyama, T., Sudo, R., 1992. Degradation of cyanobacteria, Microcystis by microflagellate, Monas-Guttula. Water Science and Technology 26 (9–11), 2173–2176. Svrcek, C., Smith, D.W., 2004. Cyanobacteria toxins and the current state of knowledge on water treatment options: a review. Journal of Environmental Engineering and Science 3 (3), 155–185. Thostrup, L., Christoffersen, K., 1999. Accumulation of microcystin in Daphnia magna feeding on toxic Microcystis. Archiv Fu¨r Hydrobiologie 145 (4), 447–467. Ueno, Y., Nagata, S., Tsutsumi, T., Hasegawa, A., Watanabe, M.F., Park, H.D., Chen, G.C., Chen, G., Yu, S.Z., 1996. Detection of microcystins, a blue–green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis 17 (6), 1317–1321. Van Donk, E., Cerbin, S., Wilken, S., Helmsing, N.R., Ptacnik, R., Verschoor, A.M., 2009. The effect of a mixotrophic chrysophyte on toxic and colony-forming cyanobacteria. Freshwater Biology 54 (9), 1843–1855. Warming, T.P., Mulderij, G., Christoffersen, K.S., 2009. Clonal variation in physiological responses of Daphnia magna to the strobilurin fungicide azoxystrobin. Environmental Toxicology and Chemistry 28 (2), 374–380. Zhang, X., Hu, H.Y., Hong, Y., Yang, J., 2008. Isolation of a Poterioochromonas capable of feeding on Microcystis aeruginosa and degrading microcystin-LR. FEMS Microbiology Letters 288 (2), 241–246. Zhang, X., Hu, H.Y., Men, Y.J., Yang, J., Christoffersen, K., 2009. Feeding characteristics of a golden alga (Poterioochromonas sp.) grazing on toxic cyanobacterium Microcystis aeruginosa. Water Research 43 (12), 2953–2960. Zhang, X.M., Watanabe, M.M., Inouye, I., 1996. Light and electron microscopy of grazing by Poterioochromonas malhamensis (Chrysophyceae) on a range of phytoplankton taxa. Journal of Phycology 32 (1), 37–46.