Review Reports - Two-Decade Changes in the Ciliate Assemblage Feeding Pattern Reflect the Reservoir Nutrient Load

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This MS deals with data from a long term monitoring (nearly 25 years) of a particular site for its ciliate composition. Ciliates are indeed a vital member of the microbial loop and may be often absent as monitoring tools for system health and change. The MS provides many data points, but the overall story and rational for the study is often muddled, and provides a text which is confusing in several places. The title provides a statement which is perhaps not adequately answered based on the MS text. The results section are jumbled and the introduction is lacking of literature citing similar studies, as well as further rational to do the study, and what areas to focus on from the available long term data. The MS would benefit from a re-write with clarity in mind, of methods with more details, and of a more sound conclusion as to what was found and why this is important (not simply results). The following are other points which should be considered:

There are difficulties with English present, especially throughout the first 100 lines. It should be read by a native speaker to improve this area in particular.

Comparisons to other long term monitoring of ciliates should be presented in the intro, with a comparison of divergence or similarities of findings from these given in the results.

The study uses various fixative methods to preserve samples for later analysis. This will inadvertently discriminate against those ciliates which do not preserve in such methods, and therefore could give a skewed view of the population without ground truthing with microscopy and or molecular sequencing. This issue must be acknowledged and addressed in the MS as a limitation. Since no photos are provided, the reader must believe the authors identifications rather than be able to examine for themselves (similar to the lacking molecular sequences for ID).

Missing from the MS is actual sampling methodology? Such as how much water was taken (volume) from how large an area, etc etc.

Section 3.1.2: Plotting chlorophyl could classify mixotrophic ciliates as planktonic prey items rather than potential predators.

The MS often mentions excluding the large and scarce ciliates form the analysis (e.g. L302). But these are important members of the microbial loop and the system. How does the data look with them including? And what is the rational for exclusion beyond them changing the data in whatever way was thought unfavorable?

L 144, which is “rare” ? Stentor is quite common.

The bullet points conclusion is perhaps not the most effective tool to present novelty. A deeper rationale for doing this study in this way should be obvious, as well as novel findings which differ or confirm those from the literature. This should not be a rehashing of the results but a takeaway from all of the data presented.

The graphs throughout look like direct outputs from an old computer program. Can these be enhanced to look nicer and provide better results? Color would be nice.

Figure 5 looks rather poor quality, and could be enhanced in several ways (capitalization, removal of commas, larger font, etc).

Comments on the Quality of English Language

There are difficulties with English present, especially throughout the first 100 lines. It should be read by a native speaker to improve this area in particular.

Comparisons to other long term monitoring of ciliates should be presented in the intro, with a comparison of divergence or similarities of findings from these given in the results.

Author Response

We are grateful for the work of the reviewers, which helped us to present our results in a better way. We have changed all graphs and added new ones, including scarce but large species. On the other hand, it made the text even longer and including the English revision, it produced so many changes in the text that the manuscript is the other. It would be very complicated to mark all the changes in the text, and I decided to copy parts of the new or drastically changed paragraphs with respective line numbering to demonstrate how we have followed the recommendation of the reviewers.

Rev#1

Reply: Thank you very much for the criticism. We have been working hard on the manuscript for quite a long time, and the possibility of finding mistakes in the presentation of the general ideas, results, and discussions has already decreased. We will try to answer and correct your comments in the text.

During the 1990s, monitoring programmes in lakes and water reservoirs also included the microbial loop components. Except for the Lake Constance studies, long-term changes in the ciliate assemblage composition have not been published until now. We suppose there are long-term data from several water bodies, such as Lake Kinneret or Lake Pavin, but they are not public; Lake Biwa long-term database is known. Detailed studies, frequently related to the PhD projects, are limited to one to two years data analysis. We would like to take the possibility and analyse our longest-known data series.

We have changed the aim of the study as follows:

Line 90-97

Our study aims to analyse two decades of ciliate monitoring in the 3-m surface/epilimnion layer of the eutrophic dimictic/monomictic reservoir Slapy (Czechia). The presented data series is the longest among those published on ciliates, which apply the same sampling and analysis method without any change. It covers the period with essential changes in nutrient load and the climate, which we divided into two segments, each spanning a decade. Such a long data series allows us to consider the most straightforward statistical methods in evaluating the annual ciliate cycle and changes in the ciliate assemblage upon changing environmental conditions.

The MS would benefit from a re-write with clarity in mind, of methods with more details, and of a more sound conclusion as to what was found and why this is important (not simply results). The following are other points which should be considered:

The manuscript was re-written

There are difficulties with English present, especially throughout the first 100 lines. It should be read by a native speaker to improve this area in particular.

Reply: We did our best re-writing the mentioned parts, and English was thoroughly checked using our “native” English-speaking colleagues.

Comparisons to other long term monitoring of ciliates should be presented in the intro, with a comparison of divergence or similarities of findings from these given in the results.

Reply: It is a pity, but as mentioned above, there are no critically revised long-term ciliate monitoring data from the epilimnion/surface layer of stratified freshwater bodies, covering over two years (almost all articles look like as a result of the PhD studies), except for the Lake Constance. If something is valid in our study, it is a decade period sampling for both segments, which could avoid overestimating the assemblage behaviour during “atypical years” (there are many of them). The best analysis was shown by Dr Helga Müller et al., but she did not put generalised conclusions while showing variability between investigated years. It is a very serious attempt because ciliate growth could be so fast and short that we cannot register or are unsure if we caught the peak. If data were published (in the books on lakes Kinneret, Pavin, Biwa), only one year or a “representative year” analysis was done. Articles such as reconsidered PEG model (Sommer et al.) or book chapters (like Posch et al.) show a general ciliate growth curve, mentioning their importance, but without any supported detailed information.

I am not young, and the ciliates are my topics (annual and spatial distribution, feeding behaviour but not deep taxonomy). However, even though there has been more information recently on shallow eutrophic lakes, deeper and stratified water bodies were not studied, or the results have not been published. If you possess more information on the topics, I would appreciate it if you could provide it. I’ve spent hours looking for it on the web, finding many of our publications and those mentioned in the Introduction.

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Undoubtedly, the database of ciliates from Lake Constance produced between 1987 and 1998 is the biggest analysed one and integrates almost all layers of the comparatively deep epilimnion [16,37]. It is still exploited to model ciliate dynamics [17,38-40]. Besides the numerous articles on ciliates mentioned above, publications of long-term studies in stratified lakes covering more than three seasons do not exist, even though microbial-ecology groups are working on the problem within the national Long Term Ecological Research (LTER) programmes: Lake Kinneret (Israel) [41], Lake Biwa (Japan) [42,43], Lake Alchichica (Mexico) [44], Římov reservoir (Czechia) [33,45], or Lake Pavin (France) [46].

Hadas, O.; Berman, T.; Malinsky-Rushansky, N.; Gal, G. Protozoa (Unicellular Zooplankton): Ciliates and Flagellates. In Lake Kinneret; Zohary, T., Sukenik, A., Berman, T., Nishri, A., Eds.; Springer Netherlands: Dordrecht, 2014; pp. 247–258 ISBN 978-94-017-8943-1.
Nishino, M. Biodiversity of Lake Biwa. In Lake Biwa: Interactions between Nature and People; Kawanabe, H., Nishino, M., Maehata, M., Eds.; Springer International Publishing: Cham, 2020; pp. 69–257 ISBN 978-3-030-16968-8.
Timoshkin, O.; et al Biodiversity of Lake Biwa: New Discoveries and Future Potential. In Annotirovannyi spisok fauny ozera Baikal i ego vodosbornogo basseina [Index of animal species inhabiting Lake Baikal and its catchment area]; Timoshkin, O., Ed.; Nauka: Novosibirsk, Russia; Vol. 1, pp. 1439–1513.
Macek, M.; Sánchez-Medina, X.; Vilaclara, G.; Lugo-Vázquez, A.; Bautista-Reyes, F.; Valdespino-Castillo, P.M. Protozooplankton. In Lake Alchichica Limnology; Alcocer, J., Ed.; Springer International Publishing: Cham, 2022; pp. 213–236 ISBN 978-3-030-79095-0.
Carrias, J.-F.; Amblard, C.; Bourdier, G.; Sime-Ngando, T. The Importance of Phagotrophic Protists in Lake Pavin. In Lake Pavin; Sime-Ngando, T., Boivin, P., Chapron, E., Jezequel, D., Meybeck, M., Eds.; Springer International Publishing: Cham, 2016; pp. 307–314 ISBN 978-3-319-39960-7.

Missing from the MS is actual sampling methodology? Such as how much water was taken (volume) from how large an area, etc etc.

Reply: Yes, we are using a strange combination of various fixatives, but according to my knowledge supported by other specialists on the environmental ciliates’ investigation, the mentioned combination is giving optimum results. In routine laboratory work, formalin-fixed samples are underestimating several ciliate taxons. Better results are obtained using Lugol´s fixation and postfixation with formalin after decolouration with thiosulphate and DAPI staining (but losing some information, e.g., on kleptoplasts and very tiny autotrophic picoplankton food). However, with direct application of concentrated Bouin after Lugol, the ciliates remained intact for an undefined time (recently, I stained 10-year-old samples with the same quantitative results but better stained). According to H. Müller workshop communications [and according to our experience], Lugol destroys selectively and at different rates some taxons within several weeks). The method has also been proven in the samples postfixed with formalin as mentioned above and again postfixed with Bouin. In contrast, formalin-fixed samples, though postfixed with Bouin, hardly serve for protargol staining. More problems with anoxic ciliates exist, but this is not the case.

QPS is tricky (with Montagnes, in public we were discussing the role of the Full Moon, and several students were appointing it), in particular in soda lakes (but we resolved the problem in another lake, too); the results depend very much on the quality of tap water, which is partly used in the washing steps of the process, but it is well known to us (even though some samples were nearly lost when the potabilization plant has changed the process). In my experience with the preparations in Canada balsam, even not perfectly stained ciliates (mainly urotrichs) can be recognised using Nomarski. Be sure all the samples were analysed by the same person – by me.

Images of found ciliates representatives for feeding behaviour groups were presented in supplementary material as Figures SX

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We obtained the integrated sample of epilimnion (0–3 m) with a plankton tube sampler at three-week intervals at five points across a transversal profile, collecting a total of 45 L of sample.

Section 3.1.2: Plotting chlorophyll could classify mixotrophic ciliates as planktonic prey items rather than potential predators.

I do accept that we have not analysed the mixotrophic ciliate species biomass chlorophyll content, which seems negligible (contrasting to Tanganyika plankton analysed years ago). Mixotrophic species use photosynthesis of the symbionts or the sequestered chloroplasts while remaining predation-dependent in nutrients such as nitrogen and phosphorus (accepted since Johannes in 60’). According to the literature and our indirect results, mixotrophic species are voracious predators of their food spectrum. Moreover, it was shown that mixotrophic species do not necessarily maintain the symbionts in optimum light conditions while the host ciliate is looking for those of their prey; the symbionts and stollen chloroplasts play the role of a screen, shading genetic information of the ciliate. We suppose that mixotrophic nanoplankton (flagellate) feeders behave much more as predators than primary producers. We put it into the Discussion.

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However, the feeding of strombidiids is not limited to nanophytoplankton, and they are efficient HNF and bacteria feeders [19,85,94,103], which explains their elevated biomass even in a lack of cryptomonads. On the other hand, though they possess mainly kleptoplasts in their cells (Figure S1), it is not feasible that their biomass contains Chl a significantly apportioning the total Chl a concentration as was observed in Lake Tanganyika [7]; other common mixotrophs with zoochlorellae like PF P. viridis or RH Lagynophrya spp., Askenasia chloreligella or Monodinium sp. (large) never reached sufficient biomass.

In the analysis case, including ciliate biomass means incorporating the large species biomass varying in the same order of magnitude as the total biomass value does not serve well. Statistically, it should not be correct. However, if we accept the fact that large species were present in some samplings, we can incorporate such data for the median-based analysis. Though the median remained zero, the order of data was changed, resulting in different median values. We explain the problem as follows:

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A special treatment had to be applied to large-cell but low-abundance species because of their very high biomass standard deviation with the median value of zero; we excluded them from the analyses of the assemblage behaviour based on the mean values such as multivariate analyses. However, their incorporation into the analyses is valid whenever the median attempt was applied because they could change the order within the sampling data. Throughout the text, we comment on the differences in the analysis without- and incorporating large-species data, but the graphs of total biomasses were put only in the supplementary material.

Moreover, all commentaries on the annual growth contain a reference to Total ciliate biomass (Figures in Supplementary material)

L 144, which is “rare” ? Stentor is quite common.

Yes, it was common even in our samples. However, it did not reach a median value higher than zero. We added the species to the graphics in the Supplementary materials, which helped to change the final median value of total ciliates. I have added all known articles on Stentor from stratified oligo-mesotrophic lakes, including one of mine

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During both periods, we observed large filtering Stentor sp. (Stentoridae) periodically in the spring phytoplankton bloom (about April) and/or during stratification (from June to August). However, its median abundance was zero. When we included Stentor sp. data (Figure S9c,d), an additional local peak appeared in the middle of stratification during period B.

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The occurrence of Stentor sp. (not included in NMDS) changed the median value of nanoplankton heterotrophic feeders before a spring phytoplankton peak during period A, while in B, another peak appeared during the stratification. However, genus Stentor data from stratified oligo-mesotrophic water bodies are scarce but from Patagonia, Argentina/Chile [92–97].

Foissner, W.; Wölfl, S. Revision of the Genus Stentor Oken (Protozoa, Ciliophora) and Description of S.Araucanus Nov. Spec, from South American Lakes. J. Plankton Res. 1994, 16, 255–289, doi:10.1093/plankt/16.3.255.
Modenutti, B.E.; Balseiro, E.G.; Moeller, R. Vertical Distribution and Resistance to Ultraviolet Radiation of a Planktonic Ciliate, Stentor Araucanus. SIL Proc. 1922-2010 1998, 26, 1636–1640, doi:10.1080/03680770.1995.11901006.
Macek, M.; Šimek, K.; Bittl, T. Conspicuous Peak of Oligotrichous Ciliates Following Winter Stratification in a Bog Lake. J. Plankton Res. 2001, 23, 353–363, doi:10.1093/plankt/23.4.353.
Woelfl, S. The Distribution of Large Mixotrophic Ciliates (Stentor) in Deep North Patagonian Lakes (Chile): First Results. Limnologica 2007, 37, 28–36, doi:10.1016/j.limno.2006.08.004.
Pucciarelli, S.; Buonanno, F.; Pellegrini, G.; Pozzi, S.; Ballarini, P.; Miceli, C. Biomonitoring of Lake Garda: Identification of Ciliate Species and Symbiotic Algae Responsible for the “Black-Spot” Bloom during the Summer of 2004. Environ. Res. 2008, 107, 194–200, doi:10.1016/j.envres.2008.02.001.
Mašín, M.; Čuperová, Z.; Hojerová, E.; Salka, I.; Grossart, H.; Koblížek, M. Distribution of Aerobic Anoxygenic Phototrophic Bacteria in Glacial Lakes of Northern Europe. Aquat. Microb. Ecol. 2012, 66, 77–86, doi:10.3354/ame01558.

We do agree. It was rewritten.

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The presented results as part of the long-term monitoring of reservoir Slapy, to which the ciliate analysis was added in 1994, becoming one of the longest from freshwater bodies. Thanks to a two-decade monitoring data there was a possibility to apply the most straightforward statistical approach of median and interquartile range. It confirmed the ciliate position in the updated PEG model, showing two prominent peaks related to spring and summer phytoplankton maxima.

The observation period was divided into two sections, which differed by the reservoir's nutrient load and were associated with different patterns. In the first period, with higher nutrient loading, the spring peak of ciliate biomass was much higher than the summer one. The phytoplankton growth suppressed the ciliates. During the lower nutrient loading period, two similarly high peaks were observed, consisting mainly of mixotrophic species. If the ciliate biomass data were time-normalised using a calculated day of stratification, a spring peak and a clear-water phase would be well defined. However, no adequate improvement was observed at the end of the stratification as the duration of the stratification varied.

It has been shown that the empirical chlorophyll a concentrations marking the beginning of the spring peak at 5 µg/L and the end of the summer peak at 7 µg/L coincide with the prominent peak of the ciliates: The spring peak of algae-hunting ciliates (Balanion planctonicum, urotrichs; more recently also Histiobalantium spp.) and heterotrophic nanoplankton-filtering tintinnids before stratification, and a peak of mixotrophic nanoplankton filtering ciliates in the stratification event, which lasted longer during the period of lower nutrient load. However, the ciliates showed a higher biomass before the summer peak of chlorophyll a. Only one ciliate, the genus Mesodinium, reached its maximum during the autumn mixing.

Using the hydrodynamic model to calculate the water age in the epilimnion/surface layer proved helpful in understanding ciliate growth there; the layer was well separated, which explains the straightforward behaviour of the ciliates and the composition assemblage changes with increasing nutrient limitation. On the other hand, no information can be extrapolated about the composition and activity of the ciliate assemblage in other parts of the water column.

By monitoring the ciliate assemblages in the surface layer for so long, we gained valuable insights into their role within the microbial loop of the plankton food web. Our findings revealed an annual periodicity and long-term variations. It is recommended that future monitoring cover additional layers, such as deep chlorophyll a maximum or oxygen local minimum, if present, to capture the complexity of water bodies such as reservoirs or lakes with continuous river flow.

The graphs throughout look like direct outputs from an old computer program. Can these be enhanced to look nicer and provide better results? Color would be nice.

Figure 5 looks rather poor quality, and could be enhanced in several ways (capitalization, removal of commas, larger font, etc).

In fact, the programme version is the last one from 2024. The mistake is mine; I was following the traditional black-and-white expression of data. I’ve redesigned everything to be in colour, the data are the same

Comments on the Quality of English Language

There are difficulties with English present, especially throughout the first 100 lines. It should be read by a native speaker to improve this area in particular.

We did our best, and using our native-English-speaking colleagues

Comparisons to other long term monitoring of ciliates should be presented in the intro, with a comparison of divergence or similarities of findings from these given in the results.

Reply: As I’ve commented above. Long-term monitoring of ciliates’ data are not published except for the data set of Lake Constance. It was pointed out in the introduction and added to the discussion

I hope we did sufficiently improve the manuscript quality, but we are open to following your recommendation to complete it on the best level.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Authors of the reviewed paper presented results of the long-term monitoring the ciliate groups in reservoir Slapy. The monitoring the ciliate assemblages in the surface layers gained valuable insights into their role within the microbial loop of the plankton food web.

The methodological level of the work is very high, results original and interesting, hence the paper may be recommended to publication as it is.

Author Response

Rev#2

Thank you very much for your positive opinion of our work. On the other hand, we thoroughly followed detailed revisions of the other revisors.

Reviewer 3 Report

Comments and Suggestions for Authors

The MS studied the changes in ciliate communities and their relationship with several environmental factors in two reservoirs from 1994 to 2018. The research has collected a large and valuable dataset. However, it lacks in-depth analysis of the data. In most cases, only temporal changes are analyzed, but it lacks necessary statistical analyses, such as the similarity of community composition over time and whether there are statistically significant differences. Simply creating time series variation graphs is not sufficient. So I suggest to further analyze the data.

Author Response

Rev#3

Thank you very much for your opinion. As your colleague was asking us for even more analyses (including large excluded species), we have analysed again the data set.

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We applied the programme Prism 10 automatic plot of medians with the interquartile range (IQR) based on raw data to construct a representative seasonal curve of the ciliates and limnology variables [59,61]. Non-metric multidimensional scaling (NMDS) with the Bray–Curtis distance measure was used to find a configuration of years in the ordination space where distances between years correspond to dissimilarity within the explanatory variables or ciliate composition. NMDS was done using Canoco 5.15 [62]. Analysis of similarity (ANOSIM) provided a non-parametric multivariate test that found potential differences between periods A and B. ANOSIM is based on test statistic R, which compares distances between groups with distances within groups. Bray-Curtis distance measure was used similarly to NMDS. ANOSIM was computed using PAST 4.17 [63].

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We characterised the years of the study using a non-metric multidimensional scaling (NMDS) based on explanatory variables composed of temperature, concentrations of dissolved oxygen, total phosphorus (with particles <40 µm), DIN, water age, chlorophyll a, and abundances of ciliate prey bacteria, HNF and Rhodomonas spp. (Figure 10a). We did not include round-the-year sampling data to the graph but those covering all principal ciliate peaks: from spring phytoplankton peak-related, i.e., two sampling dates before the event of stratification, to the end of summer phytoplankton peak with two ciliate local maxima, i.e., to the end of stratification). Only a possible winter (February–March) B. planctonicum and autumn (November) Mesodinium spp. maxima were not included.

According to the physical-chemical and ciliate-prey analysis, years from periods A and B form well-defined clusters with only small overlapping (Figure 10a). However, the same procedure with the ciliate data gave a less pronounced grouping (Figure 10b). According to vector directions, AH ciliates were driven in the same direction as MN ciliates, while HN showed a contrasting direction to them. PF and RH showed a trend similar to the ciliate assemblage biomass (corrected; without scarce and large species). ANOSIM analysis supported above-mentioned clusters and revealed significant difference between periods A and B based on explanatory variables (R = 0.51, P < 0.001) and ciliate functional groups (R = 0.29, P < 0.01).

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The NMDS analysis identifies possible causal relations between the study years using the ciliate data covering all the assemblage annual peaks (for data selection, see Chapter 3.3; Figure 10). However, the grouping of years from periods A and B did not form such distinct clusters in the graph as in the case of environmental variables and possible ciliate prey.

The analysis identifies similar vector directions of the ciliate assemblage biomass, picoplankton filtering (PF) and raptorial and flagellate hunting ciliates (RH). PF were biomass dominating or essential through the whole analysed period. Depending on the species composition, their first peak appeared before or during the stratification event, and the second passed the clear-water phase until the end of the stratification. Halteriids (Halteria grandinella, Pelagohalteria viridis) and small strobilidiids (Rimostrombidium humile, R. brachykinetum and other unidentified species) were the most common bacterivores/omnivores of the lake communities [16,19,26,28,81,83]. On the other hand, the low importance of minute scuticociliates would be surprising if we did not consider the nearly permanent DO oversaturation of the Slapy reservoir surface. At the same time, their optimum layer should be a local DO minimum [29,76]. Minute scuticociliates (Cyclidium glaucoma, Cinetochilum margaritaceum) were the most critical species; it concurs with the observation of, e.g., Müller [16]. Solitary peritrichs, mainly Vorticella aqua-dulcis and Vorticella sp. colonising diatoms Fragillaria sp., and Pseudohaplocaulus sp. on cyanobacteria Anabaena sp. were the most common within the group [26]. Recently, a new observation confirmed the omnivory of peritrichs and, in the case of pelagic free-swimming vorticellids also, efficient ingestion of nanoplanktonic, e.g., cryptomonads [88-89]. Adding pelagic colonial Epistylis spp. to the median calculation did not change the result substantially.

RH did not reach high biomass (except for scarce, e.g., Pelagodileptus sp., Lacrymaria sp. or mixotrohic large Monodinium sp., excluded from the non-parametric analysis but included in the comparative plot of the ciliate groups’ biomasses (Figure S11). Their occurrence with their food, e.g., HNF and related bacterioplankton, is logical. As in other water bodies, Askenasia sp. and Lagynophrya spp., both heterotrophic and mixotrophic, were common [19,26,90] as well as Enchelys, and minute Monodinium. Minute Mesodinium spp. biomass replaced other RH during autumn-winter mixing (not included in the NMDS analysis). Though the median from period A was diminutive, it was of the same value as the others with quite a high range in period B. Freshwater mesodinia data are scarce [91].

Heterotrophic nanoplankton filtering ciliates (HN) presented the vector direction very differently. The group dominated by tintinnids presented the biomass peak before the stratification event. However, their feeding preference should be like those of mixotrophic nanoplankton filtering ciliates (MN), culminating just at the event of stratification. Tintinnids were probably entering the surface layer of the Slapy reservoir with the water inlet. In the Římov reservoir, the tinitinnid peak was localised at the temperature of the inlet river water flow [33,64]). Rimostrombidium lacustris should have the same feeding preferences as MN [56], but the ciliate maximum was observed when Rhodomonas spp. abundances would not support the ciliate growth. Occurrence of Stentor sp. (not included in NMDS) changed the median value of nanoplankton heterotrophic feeders before a spring phytoplankton peak during period A, while in B, another peak appeared during the stratification. However, genus Stentor data from stratified oligo-mesotrophic water bodies are scarce but from Patagonia, Argentina/Chile [92–97].

The algae hunting (AH) ciliates´ vector is almost the same as that of MN. It could be related to their preferred prey, minute photosynthetic cryptomonads, including Rhodomonas spp. [56,88,90,98], but strombidiids use them mainly as a source of kleptoplasts [99-100]. On the other, the feeding group maxima occurred in different periods. AH, winter/spring peaks were composed first of B. planctonicum, which should prefer temperatures below 18 °C [101]. It was followed by Urotricha spp. and, particularly in period B, by Histiobalantium spp. during the spring phytoplankton peak [26,28,30,56] but already dropping with the stable stratification when MN reached their maximum.

After a short clear-water phase (periodically observed in one sampling date), both AH and MN could develop again, though rhodomonads did not already support their growth; Müller et al. already observed the discrepancy [90]. We suppose both groups also ate upon HNF, which were invisible in the feeding vacuoles. Ingestion of bacteria by minute prostomes (urotrichs, B. planctonicum) was negligible [19], and we never observed ingestion of them by Histiobalantium spp., as proven in cultures [90] but discarded when suitable flagellate food was present [88,102]. Contrary to the above mentioned experiments, we observed B. planctonicum at water temperatures up to 22°C during the summer phytoplankton peak.

However, I guess that our median method is reasonable for such long monitoring with only some scepticism

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Median sampling results showing the annual cycle of the ciliate behavioural groups are statistically correct, considering many observed seasons. However, it should not be absolutised because the range of data in any long-term monitored water body is vast due to “unusual years” (Figures 2, S11). The problem behind such observation is related to the “wished sampling period”, according to the ciliate generation times and monitoring programme possibilities. It was repeatedly shown that significant changes in the ciliate assemblage used to take place within a week.

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The presented results are part of the long-term monitoring of reservoir Slapy, to which the ciliate analysis was added in 1994, becoming one of the longest from freshwater bodies. Thanks to two-decade monitoring data, applying the most straightforward statistical approach of median and interquartile range was possible. It confirmed the ciliate position in the updated PEG model, showing two prominent peaks related to spring and less to summer phytoplankton maxima.

I hope we sufficiently improved the manuscript quality, but we are open to following your recommendation to complete it on the best level.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have spent considerable time rewriting the paper and addressing each point in detail in their response.

One point I would suggest is to make sure that the absence of literature on the subject, and the lack of similar studies, which were so well documented in the review report are present also in the text; as this enhances the novelty of the study.
Similarly and strengthening of the methods in the reply should appear also in the MS.

Comments on the Quality of English Language

English is fine

Author Response

Rev#1

Reply:

We put one paragraph on the fixation to the discussion

Throughout the years, one of the critical problems in the quantitative investigations of natural ciliate populations has been the use of fixatives, which altered the results, and the Utermöhl method limits the use of more powerful objectives. In routine monitoring work, formalin-fixed samples underestimate several ciliate taxons; better results are obtained using Lugol´s fixation and postfixation with formalin after decolouration with thiosulphate and DAPI staining [8,13,18–20]. QPS has solved many of these problems, and according to our literature-supported knowledge, the mentioned direct application of concentrated Bouin’s fluid after Lugol’s iodine gives the optimum results [22–24,57,67]; the ciliates remained intact for a long, undefined time. Another possible problem is connected with changes in who analysed the preparations; in our study, all the samples were analysed by the same person. However, there are discrepancies between the taxonomists and the specialists on the environmental ciliates’ investigation [58] because of, e.g., a necessity of using environment-soft fixative on the boat (such as Lugol) instead of the direct Bouin fixation.

and the other on the lack of data to be compared

The absolute biomasses of peaking ciliates in our study (interquartile range up to 30 μg/L) are lower than in shallow eutrophic lakes [67,80-82] but comparable with the Lake Constance results [16,37,38,79]. This aligns with the observed oligotrophication trend in the Slapy reservoir [49]. However, Müller et al. [16] did not state generalised conclusions while showing variability between investigated years. It is a very serious attempt because ciliate growth might be so fast and short that we could not register or were unsure if we caught the peak. In our study, the solution to the problem was to calculate medians from two-decade sampling periods, which could avoid overestimating the assemblage behaviour during “atypical years”. There are no other critically revised long-term ciliate monitoring data from the epilimnion/surface layer of stratified freshwater bodies, covering over two years. If data were published in the books (lakes Kinneret, Israel; Pavin, France; Biwa, Japan [41,42,46]), only one year or a “representative year” analysis was done. Articles such as reconsidered PEG model [1] or book chapters [83] show a general ciliate annual abundance curve, mentioning their importance, but without any supported detailed information.

Reviewer 3 Report

Comments and Suggestions for Authors

P1 L19-L26 There are too many “we"

P1 L35 L32 what does PEG mean?

P6 L191 I suggest using CCA to explore the relationship beweetn environmental factors and ciliates.

Results:

3.2 seasonality: Since the topic is about nutirent load and ciliate, I don't think the seaonality is close to this topic.

Author Response

Rev #3

P1 L19-L26 There are too many “we"

We have revised the mentioned paragraphs, and we changed the style; in several cases, we returned to passive-voice expressions.

P1 L35 L32 what does PEG mean?

The full name of the Plankton Ecology Group (PEG) model was included in the summary and the text (it is a terminus technicus for the annual plankton development in water bodies formulated by Sommer et al.)

P6 L191 I suggest using CCA to explore the relationship between environmental factors and ciliates.

CCA vs. NMDS

The suggested statistical approach using canonical correspondence analysis (CCA) is defined by Euclidean distances. CCA explicitly looks for projection rules using metric methods. On the other hand, non-metric dimensional scaling (NMDS) uses a non-metric method to reduce multidimensionality and presents straightforward plots based on dissimilarity measures. Here, we decided to measure dissimilarity using the Bray-Curtis distance, which is widely used in community ecology. NMDS based on Bray-Curtis often provides an optimal estimate of "ecological distance."

During the statistical analyses, we also tried constrained methods (RDA because of the length of gradients, not CCA) but we are convinced that our dataset fits better with non-metric scaling.

References:

Legendre, P. & Legendre, L. (2012): Numerical Ecology. Third English edition. Elsevier Science BV, Amsterdam.

Lepš, J. & Šmilauer, P. (2003): Multivariate analysis of ecological data using CANOCO. Cambridge University Press, 284 pp.

Ter Braak, C.J.F. & Šmilauer, P. (2018): Canoco reference manual and user's guide: software for ordination, version 5.1x. Microcomputer Power, Ithaca, USA, 536 pp.

Results:

3.2 seasonality: Since the topic is about nutirent load and ciliate, I don't think the seaonality is close to this topic.

Why we analysed an annual growth – seasonality- is implicitly included in the PEG model, based on former studies of nutrients’ availability in water bodies. The strength of observed phytoplankton and related organisms’ peaks (both spring and summer ones) reflect the total phosphorus concentration in the water column during a spring turnover. However, the position of the peaks and their extent varied. That is why we are analysing the annual cycle. However, such analysis is complementary to the statistical approach mentioned above, which reduces the problem to an analysis of one math of averaged seasonal dates.