Seasonal dynamics of Daphnia laevis Birge , 1878 ephippia in a tropical lake with a description of a new methodology for in situ evaluation

The effect of dormancy in zooplankton populations is still unknown, largely because of the lack of methods to estimate hatching and production of the dormant stages. This study aimed to compare the production and hatching rates of ephippia of Daphnia laevis between thermal stratification and mixing periods in Jacaré Lake (Middle Rio Doce, Minas Gerais, Brazil). For this, we collected ephippia on the sediment with core sampler and we created a device called the “Ephippial Collector”. There was a significant difference in ephippia hatching in situ between stratification and mixing periods (Pearson’s Chi-squared test p <0.001), being higher in the second one. Significant differences in the hatching rates between periods was observed in the laboratory only for ephippia collected with Ephippial Collectors (Pearson’s Chi-squared test p <0.001), being higher during the mixing period (~8%). The core sample allows the collection of a certain fraction of the sediment that may contain a mixture of ephippia produced in different periods, i.e., may contain old and not viable ephippia, which masks the hatching rate. Thus, seasonality in hatching rates of ephippia was reported only by Ephippial Collectors. The higher hatching rate observed during the mixing period in the lake suggests that individuals hatched from ephippia may contribute to the increase in the population of D. laevis in the water column at this time.


Introduction
Part of zooplankton communities may be present in the sediment of lakes as dormant stages.The dormant eggs are formed by sexual reproduction and have distinct morphology and biochemistry from those parthenogenetic eggs produced under normal conditions (Hairston Junior, 1996;Pauwels et al. 2007).Within the Anomopoda (Cladocera) order (e.g.Daphnia spp.), these dormant eggs are protected by the carapace of the mother, forming a a structure called ephippium.In general, the dormant eggs and ephippia are dark with a thick protective membrane and a well developed cytoplasm.They may remain viable for decades or centuries and accumulate in large numbers in the sediment (Hairston Junior, 1996;Cáceres, 1998;Crispim et al. 2003;Cáceres and Tessier, 2004), forming an "egg bank".Some studies have shown that fluctuations in population densities can be correlated to dormant eggs (Mnatsakanova and Polishchuk, 1996;Cáceres, 1998;Hairston Junior et al., 2000).Thus, the importance of the diapause in the zooplankton population dynamics of many species has also been increasingly recognised.
The stimuli to produce and hatch resting eggs seem to be very similar.Among the cited stimuli to produce resting eggs and ephippia, are: fluctuations in temperature (Jersabek and Schabetsberger, 1995), abrupt increases in population density (King and Snell, 1980;Ban, 1992), predation pressure (Slusarczyk, 1995;Pijanowska and Stolpe, 1996), food scarcity (Slobodkin, 1954;Gilbert, 1995), photoperiod and competition (Gilbert and Williamson, 1983), or a combination of these factors (Kleiven et al., 1992).In temperate regions, photoperiod and temperature are recognised as the main factors that stimulate the production and hatching of resting eggs (Wolf and Carvalho, 1989;Hairston Junior et al., 2000;Brendonck and De Meester, 2003;Gyllström and Hansson, 2004;Vandekerkhove et al., 2005).Other cited factors that can also affect the hatching rates are concentration of oxygen (Lutz et al., 1992;Marcus et al., 1997), salinity (Nielsen et al., 2003) and food availability (Irigoien et al., 2002).High levels of food (spring blooms in temperate environment) were seen to promote the emergence of some Cladocera species (Cáceres, 1998, Hairston Junior et al., 2000).The stimulus for hatching can also occur with a combination of factors such as photoperiod and food (Alekseev and Lampert, 2001).
The usual sampling methodologies try to estimate mainly three aspects of the dormant population: its abundance in the sediment, the production rate by the active population and the birth rate (i.e.hatching rate).Sediment samplers such as corer and dredge are used to assess the abundance and species composition of dormant eggs in the sediment.The corer-like sampler enables the collection of specific layers of the sediment, allowing us to compare ephippia stocked in different periods.Epphipial production was directly measured in lake Michigan by Kerfoot et al. (2004) using sediment traps and also by Altermatt and Ebert (2008) in rock pools using petri dishes to collect settling eggs.Hatching rate estimations of dormant eggs are usually made under laboratory conditions.Mnatsakanova and Polishchuk (1996) suggested collecting a certain layer of sediment and then incubating it under laboratory conditions similar to those of the natural environment.However, Cáceres and Tessier (2003) criticized this methodology due to the difficulty in simulating all the environmental characteristics.New methods have been proposed by some authors to measure hatching rates in situ, such as the use of hatching traps (Herzig, 1985;De Stasio 1989, 1990;Wolf and Carvalho, 1989;Cáceres 1998;Hairston Junior et al., 2000).Cáceres (1998) andHairston Junior et al. (2000) used traps placed at the bottom of the lake so that individuals hatched from epphipia in the sediment would be retained in a closed chamber collected some time later by diving.These studies have evaluated more efficiently the effect of emergence of dormant forms in the population dynamics of the studied species.
In this study we quantified and compared the production and hatching rates of Daphnia laevis ephippia between thermal stratification and mixing periods in a natural tropical lake.A new method for ephippia sampling was described and its efficiency was evaluated.

Study Area
Lake Jacaré (area: 1.03 km 2 , maximum depth: 9.8 m, coordinates: S 19° 48'38.8'',W 42° 38'55.5'') is located at the middle Rio Doce basin, near Rio Doce State Park (Parque Estadual do Rio Doce), Minas Gerais, Brazil.The region has a mesothermic tropical semi-humid climate with an average temperature of 25 °C and a marked seasonality: a rainy summer period (from September to April) and a dry winter period (from May to August) (Tundisi, 1997).The lake is surrounded by monocultures of Eucalyptus spp.and houses a fishing club, with a camping area, receiving an unknown load of domestic sewage.It has been monitored by the Program for Long Term Ecological Research (LTER/ UFMG), at first with seasonal sampling (2000 and 2001) and, from 2002 to 2010, with monthly sampling.Over ten years of monitoring, an annual circulation pattern has been observed with thermal stratification in the summer and homogenisation of the whole water column during the winter, which classifies this lake as warm monomitic.

Sampling active Daphnia laevis population in the water column
Zooplaknton samples were collected in a fixed station of the limnetic region at depths defined by the disappearance of a Secchi disk.A volume of 200L of lake water was filtered (Stihl motorised pump, model P835) through 68 μm plankton, stained with rose bengal and preserved with a 4% formaldehyde solution.All the D. laevis individuals in the sample were counted under a stereomicroscope.Samples were collected monthly from October 2007 to July 2008.Sampling frequency was increased in March and April (weekly samples), months where population densities were increasing.

Sampling ephippia in the sediment
Sediment samples for quantitative analyses of resting eggs and laboratory tests were collected (five samples) in the limnetic region with a gravity core type sampler (Kajak) on the same dates of the active D. laevis population samplings.Samples were taken bimonthly only between October 2007 and February 2008 due to the low number of active population in the water column.Just the first five centimetres of the sediment was collected, kept in dark pots and refrigerated at ± 5°C, except the one used for laboratory hatching tests, which was processed in five days at most.The water temperature profile was measured using a Yellow Spring probe (model YSI 60).

Ephippial Collector: description and sampling
The Ephippial Collectors are similar to a plankton net with a conical polyester net (used for silk screen) with 200 µm and length/mouth diameter ratio 2/1.A plastic ring with an area equal to 0.2 m 2 was used to hold the net and at the end a screwed opening cup was fixed with clamps (Figure 1).The Collectors were placed one metre above the bottom of the lake in the limnetic region suspended by a buoy and firmed by an anchor (Figure 2).Two Ephippial Collectors were placed about ten metres away from each other.These collectors remained in the lake for 22 days during the mixing period, when the active population was at its maximum.During the stratified period the collectors remained for 49 days because the active population was at its minimum and the ephippia production is very low, compared with mixing period.The difference in the number of days sampled does not interfere in the Pearson's Chi-squared Test for Count Data as this test evaluates proportions and not absolute values of ephippia collected.In addition, low proportions (as those in the stratified period) has higher variance that can only be reduced if we increase sample size (i.e.number of ephippia collected).

Quantification of eggs in the sediment and laboratory hatching tests
For quantification of ephippia, sediment samples were taken out from refrigeration, mixed with sucrose solution (1:1) and centrifuged at 3600 rpm for 5 minutes (Onbé,1978, Marcus, 1990).The ephippia in the supernatant were rinsed with distilled water and counted under a stereomicroscope (Crispim and Watanabe, 2000).Ephippia used for hatching tests were not centrifuged, but washed under a net until they were free of sediment particles.
The ephippia obtained by the Ephippial Collectors were free of sediment and were directly counted.The following ephippia classification was used: eggs with dark colouration and intact structure, without any kind of damage were considered "normal" (Figure 3); ephippia found longitudinally opened, empty or with only one embryo inside, such as those hatched in laboratory, were classified as hatched in situ; and the yellow coloured eggs, even if closed, were considered "spoiled".Hatching tests were also carried out with these spoiled ephippia.
Hatching experiments were conducted by placing a known number of ephippia in a 50ml plastic beaker containing about 20ml of filtered lake water and incubated under the same temperature recorded at the bottom of the lake (23.8 °C ± 1 °C for mixing period and 26.5 ± 1 °C for stratified period) with a 12 hour photoperiod.Eggs were examined every day for hatching success during 45 days.All born individuals and hatched ephippia were counted and removed and the water renewed.

Statistical methods
In order to verify the effect of the period (stratified/ mixing) on hatching and production rates with Ephippial Collectors, contingency tables were built and a test for homogeneity was performed using the Pearson's Chisquared Test for Count Data, with p-values estimated by the Monte Carlo technique.The statistical analysis and graphics were carried out using R software (R Development Core Team, 2010).

Results
The lake followed the thermal stratification pattern observed during the previous years of monitoring, remaining The active population of D. laevis in the water column ranged from 0 to 6076 org.m -3 , following the seasonal pattern already described for this population with higher densities in the mixing period (Figure 5) (Brandão et al., 2012).According to the number of ephippia (sum of two Collectors) produced and hatched in each period and its respective classification, a significant difference was observed between the stratified and mixing periods (Pearson's Chi-squared test p<0.001), with higher hatching rates in the second one (i.e. higher proportions of ephippia classified as hatched in situ).During the mixing period, the average daily D. laevis ephippia production, collected with the new apparatus, was 204.m -2 .day - (49.7% of the eggs classified as hatched in situ) compared with 29.m -2 .day -1 (1.3% of them classified as hatched in situ) during the stratified period (Table 1).Significant difference in hatching rate in the laboratory was observed only for the ephippia collected with Ephippial Collectors (Pearson's Chi-squared test p<0.001), with higher rates in the mixing period (~8%) (Figure 6).

Discussion
As environmental characteristics during the production of ephippia may influence its timing of dormancy (Marcus et al., 1997, Cáceres and Tessier, 2003, Cáceres et al., 2007), it is expected that the hatching rates be different between seasonal periods.This hypothesis was confirmed only with ephippia collected with Ephippial Collectors.The lack of statistical significance in the hatching rates with epphipia collected by corer is evidence that sediment can accumulate eggs produced in different periods, as the hatching rates are a mixture of the two periods.Besides that, hatching rates obtained under laboratory conditions may not represent the field situation (Cáceres and Schwalbach, 2001).They may be overestimated due to higher and constant light and lack of natural chemical inhibitors (Cáceres and Tessier, 2003) or underestimated, as a result of stable conditions, whereas the stimulus involves environmental disturbances (Panarelli et al., 2008).This could explain the low laboratory hatching rates obtained in this study (maximum 8%, with ephippia collected with Ephippial Collectors) when compared with in situ hatching rates (maximum 58%) for the mixing period.Although few studies have directly estimated hatching rates of ephippia, there is a consensus that just a small fraction of viable embryos break dormancy within a given year (De Stasio 1989, Cáceres 1998;Hairston Junior et al., 2000).However, we observed high hatching rates recorded in the Ephippial Collectors (in situ) during the mixing period (average of 50% of hatched in situ ephippia).It is known that environmental conditions determine the timing of ephippia dormancy (Marcus et al., 1997, Cáceres and Tessier, 2003, Cáceres et al., 2007) and some authors suggest that the life history of the mother and risks experienced in the water column appear to influence ephippia dormancy (Marcus et al., 1997, Cáceres andTessier, 2003).Considering the thermal stratification period as unfavourable for D. laevis, since this specie is found in low densities, the few ephippia produced remained in diapause as part of the egg bank.On the other hand, ephippia produced during the mixing period, when the environment seems to be more favourable, break dormancy in this same period, which explains the higher hatching rates.The hypothesis for it is that a high ephippia production could be a strategy for  population genetics renewal (by sexual reproduction) and renewal of the dormant egg bank.During favourable conditions, some individuals emerge, multiply, produce new dormant eggs and die after that, renewing the stock of eggs in the sediment (Crispim and Watanabe, 2000;Panarelli et al., 2008).Although high population density was a stimulus for ephippia production (King and Snell, 1980;Ban, 1992) it seems not to have inhibited hatching.Higher ephippia hatching rates occurred concomitant with higher D. laevis densities in the water column.In addition, other factors may also have induced sexual reproduction during mixing period, such as predation and competition that were not considered in this study.
It is important to note that the hatching rates obtained by the Ephippial Collectors could still not be the real hatching rates because of three factors.First, we counted just the eggs that sank, neglecting the floating eggs (Cáceres et al., 2007;Pietrzak and Slusarczyk, 2006) which should be included in the total number of eggs produced.Second, it is known that females can produce unfertilised ephippia that should not be considered in the total of produced eggs, as the fertilised and unfertilised eggs are morphologically similar and it is very difficult to distinguish them.The fact that males were recorded in a very low frequency also contributes to the question: "do the ephippia really come from sexual reproduction?".The third possible bias is the re-suspension of eggs from the sediment to the water column, but, as we noted a good correspondence between the ephippial production and the active population in the water column, this seems to be of little influence.
Our methodology, despite its simplicity, was considered suitable for this type of study.The use of Ephippial Collectors was relatively more sensitive than corer samplings and made it possible to show seasonality in both laboratory and in situ hatching rates.Better estimations of hatching are a consequence of higher selectivity, since the Ephippial Collector excludes most ephippia produced at different periods, which is itself a cause of better estimations of ephippia production rates.
stratified from October 2007 to April 2008 and mixing from May 2008 to July 2008 (Figura 4).The active D. laevis population remained in low number from October 2007 to December 2008.From February 2008 to May 2008 a pronounced increase was observed.This pattern was followed by the number of ephippia counted in the sediment core samples (Figure 5).

Figure 5 .
Figure 5. Box plot (minimum, lower quartile (Q1), median, upper quartile (Q3) and maximum) of Daphnia laevis active population in the water column and ephippia in the sediment recorded during the sampling period.The solid line is a smoothing spline of the medians.

Table 1 .
Number of ephippia and the classification recorded in situ with the Ephippial Collectors placed in lake Jacaré (MG), in stratified and mixing periods.
Figure 6.Laboratory ephippia hatching rates with ephippia obtained by corer and by collector in Lake Jacaré (MG) during the stratified and mixing periods from October 2007 to December 2008.