Protists and prokaryotes are crucial organisms involved in the cycling of matter and the biological carbon pump (Stocker 2012; Caron & Hutchins 2013; Mitra et al. 2014). To estimate their role in matter turnover, not only their biomass but also their growth rates have to be measured (Müller 1989; Leakey et al. 1992; Cleven & Königs 2007; Lai et al. 2014). There are direct, reliable methods to measure algal (that of autotrophic protists) and bacterial production under natural conditions, but assessment of protozoan production (that of heterotrophic protists) is much more difficult (Rychert 2013).
Growth rates of heterotrophic protists, which are necessary for production estimates, have long been estimated by the size fractionation method in which the organisms studied are separated from larger grazers (Verity 1986; Carrick et al. 1992; McManus 1993; Leakey et al. 1994; Nielsen & Kiørboe 1994; Macek et al. 1996; Wallberg et al. 1999; Carrias et al. 2001; Carrick 2005; Seuthe et al. 2011). In the case of ciliates, growth rates measured with this method are typically lower than 50% of those estimated from allometric equations (Leakey et al. 1994; Macek et al. 1996) that describe the relationship between cell volume, ambient temperature, and the growth rate and were formulated based on the growth of ciliates in cultures (Montagnes et al. 1988; Müller & Geller 1993). Lower rates observed during field works in size fractionation experiments are explained by food limitation of ciliate growth, but also by underestimating growth rates due to grazing between heterotrophic protists, because size fractionation does not remove predators of the same size as the organisms studied (Müller 1989; Leakey et al. 1992; Leakey et al. 1994; Macek et al. 1996; Wallberg et al. 1999; Carrias et al. 2001). Recently, Rychert (2013) and Franzé & Modigh (2013) have demonstrated that grazing among heterotrophic protists does indeed cause underestimated ciliate growth rates measured with the size fractionated method. The second problem is that ciliate growth is usually measured during a 24-hour incubation period since this includes any possible diel growth rhythms (Carrick et al. 1992; Jakobsen & Strom 2004; McManus & Santoferrara 2013) and is long enough to detect ciliate growth with the size fractionation method. However, such long incubation periods lead to the deceleration of biological processes (Carrick et al. 1992; McManus 1993; Rychert 2013), and different containment effects such as ciliate grazing on bacterial biofilm that develops on internal bottle surfaces (e.g. Macek et al. 1996). In conclusion, more field research on ciliate growth rates are required and they should be performed with methods better than size fractionation.
A method that completely removes the grazing pressure exerted on ciliates and allows a short incubation period is the modified dilution method (Rychert 2013; see also Franzé & Modigh 2013). It consists of the whole-community manipulation by creating a gradient of dilutions of whole water with a 10-μm filtrate. Through the dilution, grazing pressure decreases toward zero, and the apparent growth rate approximates the specific growth rate. Because of the filtrate used, the method allows to study the specific growth rates of protists larger than 10 μm (e.g. ciliates) that feed on prey smaller than 10 μm (nanoflagellates, small algae, or bacteria). Modifications of the dilution method (Landry & Hassett 1982) were previously applied to measure the growth rates of pelagic heterotrophic nanoflagellates (Landry et al. 1984; Berglund et al. 2005; Dupuy et al. 2007) and interstitial ciliates (Cleven & Königs 2007).
In this study, the measurements of the growth rates of common pelagic ciliates were performed with the modified dilution method (Rychert 2013) and growth rates were compared with literature data and also with rates estimated from an allometric equation formulated by Müller & Geller (1993). Experimental incubation periods were shortened to approximately 4 hours to minimize artifacts. Separate measurements performed around noon and midnight allowed the observation of diel rhythms in ciliate growth. It is still unresolved whether ciliates are food-limited in different systems and whether their growth rates are indeed lower than those in cultures. Food limitation is difficult to study, because resources are typically distributed in patches and ciliates can exploit them and be satiated even when the mean abundance of food particles calculated for larger volumes is below the saturation level (Paffenhöfer et al. 2007). In this study, the mean concentrations of food resources of the organisms in question were analyzed and discussed in order to check whether high growth rates were accompanied by saturation levels of food resources or whether the organisms exploited unevenly distributed food and were satiated despite mean concentrations of food resources that were below the saturation level. It was hypothesized that the ciliate growth in the highly eutrophic lake was as high as in cultures.
The studies were performed in large (24.7 km2), shallow (mean depth: 1.2 m), brackish (mean salinity: 0.6–0.7 PSU, Trojanowski & Antonowicz 2011, Rychert et al. 2012, Wielgat-Rychert et al. 2015) Lake Gardno (54°39’N, 17°07’E), at an offshore station in the southern part of the lake representing an open water area (Ficek & Wielgat-Rychert 2009). The lake is highly eutrophic with high average chlorophyll concentrations in the growing season (87 μg l-1) and high annual integrals of primary production at 402–471 g C m-2 (Wielgat-Rychert et al. 2010). The mean annual ciliate biomass is 107–115 μg C l-1 (Rychert et al. 2012), which corresponds to about 100 cells ml-1. In this study, the experiments were carried out in September 2011, June 2012, and June 2013. Each time, two experiments were performed (around noon and around midnight) for a total of six experiments (Table 1). Water for the experiments was taken from the well-oxygenated subsurface layer, and temperature, salinity, bacterial and flagellate abundances, and chlorophyll
Before each experiment, all the equipment was acid-washed and thoroughly rinsed with deionized water. Lake water for the experiments was prescreened through 100-μm mesh nylon gauze to remove larger zooplankton that could not be distributed representatively in the treatments. This water was treated as whole water and was carefully mixed. To produce the 10-μm filtrate, part of the water was sequentially gravity-filtered through 25-μm mesh gauze, and then twice through 10μm mesh gauze. To prevent clogging, the first filtration with 10-μm mesh gauze was carried out through three parallel filtration sets. Next, filtrates were combined and filtered through the fourth filtration set. Samples were taken from the 10-μm filtrate to assess the impact of filtration on bacterial abundance, flagellate abundance, and chlorophyll concentration. Subsequently, whole water and 10-μm filtrate were used to prepare the dilutions, in which fractions of whole water were 20% (in triplicate), 40%, 60%, 80%, and 100% (undiluted whole water, in triplicate). All dilutions were prepared in 120-ml bottles (9 in total), and each bottle was gently mixed by rotating it 50 times. Two bottles, one each for 20% and 100% dilutions, were used to assess changes in bacterial and flagellate abundances as well as chlorophyll
Environmental conditions and food resources during experiments carried out in highly eutrophic Lake Gardno. Mean values for the incubation of whole water and 20% dilution were calculated for bacterial and flagellate abundances (see Materials and Methods). In the case of chlorophyll
Experiment | Temp.(°C) | Salinity(PSU) | Ciliates(cells ml1) | Chlorophyll |
Bacteria (106 cells ml1) | Nanoflagellates (103 cells ml1) | ||||
---|---|---|---|---|---|---|---|---|---|---|
whole water | 10-μm filtrate | whole water | 20% dilution | whole water | 20% dilution | |||||
21–22/09/2011 | midnight | 15.6 | 0.59 | 67.3 | 60.9 | 7.03 | 7.04 | 5.22 | 18.7 | 11.2 |
22/09/2011 | noon | 15.4 | 0.67 | 42.2 | 61.9 | 6.28 | 5.89 | 3.26 | 19.3 | 16.9 |
13/06/2012 | noon | 20.0 | 0.92 | 118 | 50.3 | 9.17 | 8.76 | 7.87 | 20.3 | 14.6 |
13–14/06/2012 | midnight | 21.1 | 0.84 | 119 | 33.8 | 6.89 | 9.76 | 9.14 | 16.0 | 12.8 |
28–29/06/2013 | midnight | 18.4 | 0.17 | 307 | 45.3 | 7.58 | 16.4 | 14.9 | 18.1 | 15.5 |
29/06/2013 | noon | 18.2 | 0.18 | 423 | 46.8 | 8.69 | 12.7 | 10.7 | 16.6 | 15.4 |
The ciliates were analyzed in samples fixed with acid Lugol’s solution (final concentration 0.5%). The samples were stored in the dark at 4°C until they were analyzed using the Utermöhl (1958) method. The observations were conducted using an Olympus CKX41 inverted microscope. The ciliates were identified according to Foissner & Berger (1996). It was crucial to analyze the entire bottom of the Utermöhl chamber and not half of it to minimize the error caused by the uneven distribution of specimens on the bottom. Samples were dense with algae and detritus, so small volumes of water were taken for analyses: 3 ml or 10 ml for the more diluted treatments. If necessary, additional counts were performed to gather a sufficient number of specimens. In each experiment, the entire ciliate community was analyzed in one initial sample collected from whole water (see abundance values in Table 1). In the remaining samples, only two ciliate species and their growth rates were analyzed. The ciliate species were selected according to (i) high abundance, which allowed for an accurate assessment of changes in the abundance during incubation, and (ii) distinct morphology, which facilitated the identification of all specimens present in samples fixed with Lugol’s solution. Because fixation with Lugol’s solution obscures taxonomic features, the ciliates were identified to the genus level. During incubation, no evident changes in ciliate cell volumes were observed. For each dilution, the apparent growth rate (k, h-1) was calculated assuming the exponential growth during incubation:
where N1 was the final abundance (cells ml-1), N0 was the initial abundance (cells ml-1), and t was the duration of incubation (h).
The experiments were analyzed according to Landry & Hassett (1982). The encounter rates between predators and prey were gradually reduced along the dilution gradient from undiluted to the most diluted water. Thus, the apparent growth rate (k, h-1) in each dilution depended on the specific growth rate (μ, h-1), i.e. the intrinsic rate of increase, grazing pressure (g), and the fraction of whole water in the dilution (D, %):
Plotting the apparent growth rates against fractions of whole water in dilutions enabled the estimation of specific growth rates (μ, h-1) for the theoretically complete relaxation of grazing pressure (D = 0%, y-intercept, Fig. 1). A single measurement comprised 7 experimental treatments and the analysis of 15 samples (7 initial and 7 final samples, and also one additional environmental sample); thus, the growth rate estimates were statistically significant. Growth rates were measured during short incubations (3.5–4.8 h), therefore they were expressed per hour.
Measurements of ciliate growth rates were compared with potential (maximum) growth rates estimated from an allometric equation formulated on the basis of ciliate growth in cultures. The model published by Müller & Geller (1993) was chosen because it is based on the largest data set and is considered to be the best (Macek et al. 1996, Montagnes 1996). In this model, potential specific growth rates (μ, d-1) were estimated from the mean cell volume of the organism studied (V, μm3) and the ambient temperature (T, °C):
To calculate the volume of organisms studied, they were measured in samples of the whole water collected before incubation. Measurements were carried out with an ocular micrometer or image analysis software. The volume (V, pm3) of
The volume of ciliates can shrink after fixation with Lugol’s solution. This shrinkage is species-specific and also depends on the physiological state of the cells (Choi & Stoecker 1989; Wiackowski et al. 1994). Thus, no correction was applied to avoid introducing additional error. The potential specific growth rates computed (μ, d-1) were divided by 24 to calculate values per hour. Growth rate estimation for tintinnid ciliates
Next, the carbon content was calculated back to the volume using the coefficient 0.11 pgC μm-3 (Turley et al. 1986) to calculate the surrogate cell volume, i.e. volume of hypothetical protoplast containing the organic carbon allocated for production of both protoplast and lorica. This surrogate cell volume was used to calculate the potential growth rate according to the formula by Müller & Geller (1993), similarly as with aloricate ciliates.
Additionally, the tintinnid ciliate growth (μ, d-1) was calculated based on the lorica oral diameter (LOD, μm) according to the formula by Dolan (2010), which was further developed by Montagnes (2013):
The estimated growth rates (d-1) were recalculated per hour.
During all six experiments, I assessed the impact of dilution on food resources of the organisms studied in the whole water and in 20% dilution. Three parameters were studied: bacterial abundance, flagellate (ANF and HNF, size: 2–10 μm) abundance, and chlorophyll
The quantity of food resources was analyzed in each experiment separately according to the food demands of the organism in question. When necessary, they were recalculated into carbon units. The nutritional value of algae was calculated from chlorophyll
Four common ciliates were studied. In September 2011, I measured the growth rates of
As already mentioned, the growth rates of
In conclusion, the growth rates measured in this study corresponded well with the maximum values reported in the literature at similar temperatures or were even higher. Therefore, the hypothesis posed in the Introduction was confirmed. As mentioned in the Introduction, ciliate growth could be approximated with the allometric equation by Müller & Geller (1993). However, it does not necessarily estimate the maximum growth rates of particular species, but approximate the “general ciliate growth” and are especially useful for estimating the growth rates of multi-species ciliate communities (Montagnes 1996). To perform a general comparison (Fig. 2), the mean diel growth rates from measurements were compared with growth rates estimated with the allometric formula according to Müller & Geller (1993). This comparison also indicated that the growth rates measured are at the same level as rates expected from the growth observed in cultures.
Unsuccessful experiments with
The dilution method is based on the two main assumptions: (i) the growth of the organisms studied is not food-limited in subsequent dilutions and (ii) grazing pressure depends linearly on the dilution factor, which is proportional to the concentration predators (Landry & Hassett 1982; Calbet & Saiz 2013). The first assumption holds if the experimental procedure does not cause a reduction in the food resources in dilutions or, alternatively, organisms are food-satiated, both in the whole water and in the dilutions, despite the reduced food resources in dilutions. Filtration excluded bacteria attached to detritus particles. In the 20% dilution, bacterial abundance was 55–94% (mean: 81%, Table 1) of that observed in whole water. However, neither attached bacteria nor detritus particles larger than 10 μm are edible for the organisms studied. Similarly, the abundance of flagellates was also reduced. Their abundance in 20% dilution was 60–93% (mean: 80%, Table 1) of that observed in whole water. Filtration had a pronounced effect on chlorophyll
The second assumption of the dilution method is that grazing pressure depends linearly on the dilution factor. Predatory ciliates, which could prey on the organisms studied, were observed during all the experiments. Depending on the occasion, their abundance in whole water ranged from 2.7 to 13.0 cells ml-1. During all the experiments, I observed many specimens from the genera
As illustrated in Fig. 1, the apparent growth rates observed in whole water (dilution 100%) were typically negative. One could expect values close to zero, that is, balanced growth and mortality rates. At the end of each experiment, an additional environmental sample was taken to compare changes in the whole experimental water with that at the study site. In 6 out of 8 cases, apparent growth rates in whole water in bottles were lower than in the environment; however, the difference was not statistically significant (Wilcoxon’s signed rank test). Theoretically, negative growth rates in less diluted water could be caused by trophic cascade effects, because whole water was pre-screened through 100-μm mesh nylon gauze and during incubation metazooplankton smaller than 100 μm was released from grazing pressure and could graze more strongly on ciliates (e.g. Klaas et al. 2008; First et al. 2009; Calbet & Saiz 2013). This would result in the overestimated slope of the grazing curve and grazing rates. However, it is of lesser importance in this study, because only growth rates and not grazing rates were taken into account. In most cases, the specific growth rates estimated (Fig. 1, y-intercept) were comparable to apparent growth rates observed in the 20% dilution.
The dilution method relaxes grazing pressure, but does not remove other mortality agents such as (i) viral lysis (McManus 1993), (ii) toxins excreted by other protists (Verity 1986, Leakey et al. 1994), or (iii) parasites (Verity 1986; Coats & Bachvaroff 2013; Coats et al. 2014). However, these mortality agents seem to be far less important than grazing, because successful elimination of grazing pressure generally results in very high growth rates close to the intrinsic rate of increase (e.g. Nielsen & Kiørboe 1994; Franzé & Lavrentyev 2014; this study).
The number of the studied ciliate species was rather low, but their statistically significant growth rates measured under natural conditions in the highly eutrophic lake corresponded well with the rates observed in cultures. Similarly, very high ciliate growth rates in eutrophic waters are also reported by Lavrentyev et al. (2004). Thus, the growth of ciliates in eutrophic waters is as fast as in the cultures and the hypothesis put forward in the Introduction was confirmed. It should be emphasized that the isolated measurements indicate that even in less nutrient-enriched systems some ciliates can grow with rates similar to or even higher than those estimated with allometric equations, e.g. in oligo-mesotrophic Lake Pavin (Carrias et al. 2001), oligo-mesotrophic Lake Michigan (Carrick 2005), meso-eutrophic Lake Constance (Müller 1989), and in sea waters (Nielsen & Kiørboe 1994; Rychert 2013; Franzé & Lavrentyev 2014). Confirmation of the hypothesis implies that higher ciliate growth rates should be applied in ecological models (e.g. Buitenhuis et al. 2010).
The mean concentrations of food particles available for
The dilution method was confirmed to be useful for studying growth rates of pelagic ciliates during short incubations. Short incubation periods, lasting only a few hours, minimized artifacts and enabled the detection of diel rhythms in growth rates (e.g.