Feeding response of Daphnia cf . similis to different concentration gradients of Microcystis and its implication for preventing algal blooming

Daphnia are important components of zooplankton communities in lakes, ponds and reservoirs. Currently freshwater ecosystems are affected worldwide by Cyanobacterial blooms through the process of eutrophication. The objective of this study was to provide experimental evidence to the response of Daphnia to various concentrations of Microcystis. The experiment contained four treatments and two controls each with three replicates. The first control contained D. cf. similis without Microcystis and the second control contained Microcystis without D.cf. similis. The remaining four treatments contained both D. cf. similis and Microcystis at different concentration of Microcystis.. The results showed a significant negative relationships between D. cf. Similis and Microcystis across the treatments (F=294.5; p<0.00). From the four Microcystis concentration gradients, we found mortality of D. cf. Similis in HMC (high Microcystis concentration) and 67% of the original HMC gradient while in the other treatments, in 43% and 22% original HMC treatment, D. cf. Similis reproduce and attain high density. Therefore, from these results it is concluded that D. cf. similis can control the growth of Microcystis if the concentration is low but they cannot reduce an already existing bloom.


INTRODUCTION
Understanding, managing and learning about freshwater ecosystems has become increasingly significant throughout the world as the development of land continues to expand and as knowledge of the impact increases.Due to the frequency of algal blooms in the world, particularly as a result of intense, hot summers, there has been an increasing awareness of the associated issues including the creation of anoxic conditions, and risk of intoxication for those exposed to toxic cyanobacteria (Codded et al., 2005).Freshwaters such as lakes, rivers and reservoirs are the most important resources, especially in the tropics, where they are often viewed as highly productive biological systems.In Tigray Regional State, there are more than 70 reservoirs (Tsehaye et al., 2007;Tadesse et al., 2008).They provide water for fishing, irrigation and a variety of other domestic and agricultural purposes.But currently these freshwater reservoirs are affected by Cyanobacterial blooms as a result of eutrophication and global warming (Tsehaye et al., 2007;Tadesse et al., 2008).Cyanobacteria blooms have a tremendous Minalu, B and Tadesse, D (MEJS) Volume 7( 1 impact on the socio-economic and ecological values of freshwater bodies by threatening human and ecosystem health (Anderson, 2012).Prevention of this bloom is better than curing since there is a possibility for harmful metabolites to be leached that are potentially dangerous to human health.Grazer-mediated bloom control is among the different methods that are used for controlling blooms, Daphnia is an excellent candidate for this task, because it is a natural component of freshwater ecosystems, ubiquitously present and since there are strong grazers.
In the absence of fish predation, the biomass of large species of Daphnia increases and the biomass of filamentous cyanobacteria often decreases (Paterson et al., 2002).Some studies have provided evidence for ingestion of cyanobacteria by Daphnia and suggested that grazing can provide a control mechanism for cyanobacterial (Microcystis) blooms (Boon et al., 1994), or that cyanobacteria can be a complementary resource for zooplankton (Daphnia) (Kurmayer, 2001).
On the contrary several studies have shown a different scenario where Daphnia are not good grazers of cyanobacteria compared to other algal species, highlighting their insufficiency to control Microcystis proliferation (Lurlingr, 2003;Ghadouani et al., 2004).
Experiments showing that Daphnia can suppress cyanobacteria in eutrophic lakes have been limited to cases where Daphnia were able to achieve high densities before cyanobacteria became dominant (Lurlingr, 2003).Thus, it is not clear whether Daphnia can overcome the inhibitory effects of high cyanobacterial abundance on their competitive ability and invade a cyanobacteriadominated assemblage.
An increasing number of studies report tolerance of Daphnia clones to toxic Microcystis (Boon et al., 1994;Sernelle and Wilson, 2005).Gustafsson et al. (2005) conducted a study which exposed D. magna individuals to toxic Microcystis and they found that Daphnia were able to develop and pass the defense mechanisms to their offspring.From such study it was concluded from the difference in the time taken to reach maturity and smaller numbers of offspring per clutch between individuals previously exposed and those that were not.This means Daphnia has the ability to adapt to environmental conditions by passing information through maternal effects (Gustafsson et al., 2005).Similarly, Sarnelle et al. (2010) concluded that Daphnia populations with prior experience with toxic cyanobacteria show positive population growth even at high concentrations of cyanobacterial toxins.
However, interactions between bloom forming cyanobacteria and Daphnia are controversial issue in literatures (Lurlingr, 2003;Sarnelle et al. 2010).Many studies reported that a decline in biomass of Daphnia as a result of Microcystis (DeMott et al, 1999;Paterson et al., 2002;Sarnelle et al., 2010).Study on this controversial issue is important to understand the interaction between Daphnia and bloom forming cyanobacteria.Therefore, this study intends to look on the effect of D. cf.similis grazing potential on Microcystis at different concentration gradients of Microcystis.

Culturing of D. cf. similis
Daphnia samples were collected from Adi Abagie reservoir, located in Eastern Tigray, using 64μm mesh size in November, 2011.D. cf.similis were isolated from the sample and were transferred individually into plastic jars using a pipette with a large tip opening.The mothers producing the experimental animals were cultured individually in 250 ml jars of spring water supplemented with Scenedesmes, brought from KU Leuven Aquatic Laboratory, Belgium.Every two days interval the water was changed and Scenedesmes added as food.First we prepare the new water in a new jar then we transfer the Daphnia after the Scenedesmes added as food.D. cf.
similis were cultured for two generations before using them in the experiment according to the method recommended by Sarnelle and Wilson (2005).This is crucial since it gets rid of maternal effects of previous exposure and also ensures enough representation of individuals for the experiment.

Microcystis Collection and Dilution
Microcystis were collected from Gereb Mihiz, one of the reservoirs located in southern Tigray.
We were lucky to get bloom of Microcystis, thus we did not do further process of multiplication for Microcystis.Different concentration levels of Microcystis were prepared by adding different milliliters (100mL, 50mL, 25mL and 12.5mL) of concentrated sample from the reservoir into 1 liter of spring water from the initial source.The starting sample, which we refer as high Microcystis concentration (HMC), was our original sample collected from Gereb Mihiz dam which was further concentrated by repeatedly filtering it with 30µm sieve and the colonies were counted and it was 103,860 colony/l.The first sample was 900ml of HMC, the second sample consisted of 50ml of HMC and 950ml of water, which when we count the colony was found to consist 69,600 colony/l, hence the colony count was 67% of the original sample, thus it is referred as 67% of the original HMC.Dilution for the third sample was done by adding 25ml of HMC and 975ml of water and the fourth sample consisted of 12.5ml of HMC and 987.5ml water.The colony counts for the third and fourth samples were 44,600 colony of Microcystis per liter (43% HMC) and 23,000 colony of Microcystis per liter (22% of the original HMC) respectively.

Experimental Design, Sampling Method and Analysis
The experiment comprised of four treatment groups and two control groups [D.cf.similis without Microcystis and Microcystis without D. cf.similis] each with three replicates.As the experiment is done in 1000ml of sample of phytoplankton, 20 individuals (as the volume of water is only 1 liter greater than this after reproduction will create crowded condition) of D. cf.
similis were added into Microcystis containing 1 liter spring water with four different concentrations gradients (HMC, 67% HMC, 43% HMC, and 22% HMC).The purpose was to test whether D. cf.similis can invade Microcystis dominated plankton community or they fail to graze on Microcystis.
Jars (modified 2 liter plastic bags) were sampled in three days interval for 16 days.We measured dissolved oxygen, pH, temperature and conductivity in situ with a WTW Multi 340 I electrode.
Turbidity and concentration of chlorophyll was measured using fluorometer (Turner Aquafluor; average of three measurements).D. cf.similis were directly counted using a pipette with a large tip opening at 0 and 4 days, but as the number of D. cf.similis increased with time (8-16 days) for 43% HMC, 22% HMC treatment and control, 100ml sub samples were taken and filtered using 64µm mesh size and the filtrate was preserved in 10% sugar saturated formalin.Then individuals were counted using stereomicroscope according to the method recommended in Fernando (2002).Five ml of Microcystis sample was taken with a pipette and preserved by lugol's solution and colonies were counted using inverted microscope according to the method used in John et al. (2002).
Results from the experiment were statistically analyzed with repeated-measures ANOVA and student t-test using SPSS version 16.Repeated measures analysis provides tests of overall treatment.Association between D. cf.similis and Microcystis at each treatment was analyzed with paired t-test at 0.05 significant levels.

RESULTS
The comparisons of the colony counts of Microcystis and individual survival counts of D. cf.
similis on the start of the experiment, day zero, and end of the experiment, day 16 across the concentration gradients showed variation among the different concentration gradients (Tables 1).Microcystis concentration gradients had effect on growth, reproduction and survival of D. cf.
similis compared to that of the control with no Microcystis (which contain only Scenedesmus as food).Repeated measure of ANOVA revealed significant treatment -time interaction for D. cf.
similis and Microcystis (F=294.5;p<0.000).Analysis using paired t-test also revealed the negative correlation between D. cf.similis and Microcystis across the treatment along time (p< 0.05).
In addition to the comparisons of the colony count of Microcystis and survival of D. cf.similis across the concentration gradients of Microcystis, percentage population growth of D. cf.similis relative to control was compared to assess the extent at which D. cf.similis potential to control Microcystis (Tables 2 and 3).Microcystis showed significant differences between the controls (with no D. cf.similis) and the two treatments (43% HMC and 22% HMC).Density of Microcystis remains high in the control with no D. cf.similis but significantly decreased in 22% and 43% HMC treatment (Table 3).

Interactions between Microcystis and D. cf. similis
HMC treatment contains the highest concentration of Microcystis which is more than 103,860 colonies per liters and 20 individual of D. cf.similis.However, at the end of the experiment all D. cf.similis died.In the second treatment, 67% HMC (69,600 colonies), 80% of D. cf.similis died (Table 2).
The response of Microcystis with D.cf.similis at HMC and 67%HMC treatment is depicted in figure 1.   justified by the fact that cyanobacteria (Microcystis) are among the complementary food items of Daphnia (Kurmayer, 2001).There are also other supporting evidences that indicate ingestion of Microcystis by Daphnia species (Rohlack et al., 2001).This indicates that D. cf.similis was able to feed on Microcystis and result in decrease in the density of Microcystis in lower and medium treatments.This is in agreement with the finding of Paterson et al. (2002)  The controlling effect of D. cf.similis on Microcystis is controversial (Lurlingr, 2003;Sarnelle et al., 2010).However, the result of the present study also clearly showed two important conditions.
This condition was also reported by Sarnelle (2007) who conducted enclosure experiment using Daphnia pulicaria and Microcystis at different initial conditions.
The growth and reproduction of D. cf.similis in the treatments was not the same as that of the control which had no Microcystis.This is due to the fact that Microcystis are not good in nutrition because they lack essential fatty acids or lipids, and the poor nutritional value may have effects on Daphnia growth and reproduction (Von Elert et al., 2003).Probably this may be the reason for low density of D. cf.similis in 22% HMC and 43% HMC even though they can survive and reproduce in the presence of Microcystis.Significant decrease in Microcystis colony in 43% and 22% HMC treatment was primarily due to D. cf.similis.Tadesse et al. (2009) also found negative association between Daphnia carinata and cyanobacteria and reported a drop in the relative abundance of cyanobacteria along with an increase in the densities of D. carinata.
The result of this study clearly showed how a gradient of Microcystis concentration affects the ability of Daphnia to suppress Microcystis.This is in agreement with the majority of studies that reported the effect of Microcystis on Daphnia (Lurlingr, 2003;Ghadouni et al., 2004).This is in agreement with Reinikainen et al. (1994) who reported higher mortality of Daphnia when Microcystis is abundant or when it is the only food source.Besides, the colonial forms of Microcystis cells have digestibility, toxicity and low fatty acid composition (Gliwicz and lampert, 1990;Sarnelle et al., 2010;Muller, 1995).The other possible explanation could also be due to the increased secondary metabolites produced by Microcystis, such as poly unsaturated fatty acid or protease inhibitors might result for the death of Daphnia (Watanabe et al., 1988;DeMott et al., 1991).It may be also true in case of this study because high mortality of D. cf.
similis was observed at higher concentration of Microcystis.Some studies reported that the ability of Daphnia to tolerate or resist the toxic effect of Microcystis (Sarnelle and Wilson, 2005).However, to tolerate the toxic effect of Microcystis, Daphnia need long time exposure to toxic, however, our experiment was performed in short period of time and the HMC treatment contained extremely higher concentrations of Microcystis which might be the reason for the death of D. cf.similis in this treatment.
In addition, the toxic effect of Microcystis, colony size also affects the survival probability of D.
cf. similis.Even though the size of Daphnia is enough to feed on colony (Rohrlack et al., 2001), at HMC and 67% HMC treatments, there were attachment of many colonies into one large colony and the movement of D. cf.similis was highly affected.All D. cf.similis remain in the bottom side of the jar and the long chain of Microcystis did not even allow them to move.Due to this they were not active in their movement and spend most of their time at the bottom of the jars.This effect of Microcystis was also observed in the work of Debenardi and Guissani (1990), who found the morphological effect of Microcystis on Daphnia.Concentration gradients of Microcystis had great effect on the collection of available food (Gliwicz and Lampert, 1990).As the concentration of Microcystis increase their mechanical interference with the collection of available food sources also increase.Therefore, at higher concentration of Microcystis, Daphnia may not be able to filter the available food and this may lead to starvation then death followed.
In general HMC and 67% HMC treatments produce stressed environment and all this are the probable reason for the death of D. cf.similis before the experiment ended.Due to all these reasons D.cf.similis were unable to reproduce and grow.This study indicated that Microcystis had lethal effects upon Daphnia if the concentration of Microcystis was very high.However, although it was not possible to distinguish whether the Microcystis was toxic or non-toxic in this study, previous study in Tigray reservoirs has shown that they could be potentially toxic (Tsehaye, 2009).
Generally the result showed a negative association between D. cf.similis and Microcystis across the treatment and found that Microcystis concentration gradient had strong effect on the interaction of D. cf.similis and Microcystis.Depending on the concentration gradients of ):3-15, 2015 © CNCS, Mekelle University 4 ISSN: 2220-184X

Figure 2 .
Figure 2. Interactions of D. cf.similis and Mycrocystis at different concentrations gradient of Mycrocystis (Log transformed count per liter).

Figure 3 .
Figure 3.The survival (existence) of Dcf.Simils (log transformed individual per liter) along the concentration gradient of Microcystis.

Table 1 .
Comparison of the average count of D. cf.similis and Microcystis at day 0 and at the end of experiment (16 day) across the concentration gradient of Microcystis.Note: * 1 The Control (with no Microcystis) was supplemented with Scenedesmes.
* 2 The nutrient source for the control (with no D. cf.similis) is dam water.

Table 3 .
Microcystis treatment D. cf.similis did not attain this density.At low concentration of Microcystis (22% HMC) the density of D. cf.similis reached high (155 individuals per liter) compared to the 67% HMC (4 individuals) and 43% HMC (86 individuals).Percentage of Microcystis left at the end of experiment relative to day zero.
At the highest concentration of Microcystis, all D. cf.similis died while in the other Microcystis treatments (43% and 22% HMC) D. cf.similis grow and reproduce and able to control the Microcystis until the Microcystis remain (with no Microcystis) group and the other treatments.At the end of the experiment maximum density of D. cf.similis (302 individuals per liter) was recorded in the control with no who provide considerable evidence that Daphnia can have large negative effects on cyanobacterial abundance despite the relative grazing-resistance.The twenty D. cf.similis that were immersed at lower (22% of the original HMC) and medium (43% HMC) concentration of Microcystis managed to survive, grow, reproduce and create negative impact on the Microcystis population growth.This finding is in agreement with the work of Chen et al. (2007), who observed a reduction of Microcystis population as a result of Daphnia stocking.