**Changes in Planktivory and Herbivory Regimes in a Shallow South American Lake (Lake Blanca Chica, Argentina) Over the Last 250 Years**

#### **David Carrozzo 1, Simona Musazzi 2, Andrea Lami 2, Francisco E. Córdoba <sup>3</sup> and María de los Ángeles González Sagrario 4,\***


Received: 24 December 2019; Accepted: 18 February 2020; Published: 22 February 2020

**Abstract:** Shallow lakes are vulnerable ecosystems impacted by human activities and climate change. The Cladocera occupy a central role in food webs and are an excellent paleoecological indicator of food web structure and trophic status. We conducted a paleolimnological study in Lake Blanca Chica (Argentina) to detect changes on the planktivory and herbivory regimes over the last 250 years. Generalized additive models were fitted to the time series of fish predation indicators (ephippial abundance and size, mucrone size, fish scales, and the planktivory index) and pheophorbide *a* concentration. The cladoceran assemblage changed from littoral-benthic to pelagic species dominance and zooplankton switched from large-bodied (*Daphnia*) to small-bodied grazers (*Bosmina*) ca. 1900 due to increased predation. The shift in planktivory regime (ca. 1920–1930), indicated by fish scales and the planktivory index, as well as herbivory (ca. 1920–1950), was triggered by eutrophication. Changes in planktivory affected the size structure of *Bosmina*, reducing its body size. This study describes the baseline for the lake as well as the profound changes in the composition and size structure of the zooplankton community due to increased predation and the shift in the planktivory regime. These findings will provide a reference status for future management strategies of this ecosystem.

**Keywords:** *Daphnia*; *Bosmina*; pheophorbide *a*; fish predation; grazing; ephippia; cladocera sub-fossil remains

#### **1. Introduction**

Zooplankton play a pivotal role in aquatic ecosystems and global biogeochemical cycles. In fact, zooplankton act as a hinge in the aquatic food web because they exert a control role on phytoplankton through grazing (performed by herbivorous filter organisms such as cladocerans and rotifers) and are the food resource of higher trophic levels [1,2]. Zooplankton are highly sensitive to changes in aquatic ecosystems [3]. The effects of environmental disturbances can be detected through changes in species composition, abundance, and body size distribution. Grazing capacity on algae is directly related to the body size of organisms—the larger size, the greater grazing capacity [4]. Zooplankton are sensitive to the predation pressure exerted by fish (decreasing their body size) and macroinvertebrates (increasing their body size [5]). Jeppesen et al. [6] showed that the top-down control in shallow lakes

is stronger due to the lower abundance of piscivorous relative to planktivorous and omnivorous fish. Cladocerans are an important component of the zooplankton and the chitinous structures of their exoskeleton (postabdomen, jaws, claws, antenna segments) and resistance eggs (ephippia) are an important, well-preserved, and taxonomically well-known autochthonous element of lake sediment. Due to their potential role as indicators of various environmental conditions, the study of subfossil remains of Cladocera has been very useful in identifying and inferring changes in trophic status, predation by fish and invertebrates, macrophyte coverage, as well as chemical and physical properties of lake water [7–9].

Shallow lakes are considered as an ecosystem model in which regime shift, that is, rapid and abrupt transition from one persistent regime to a different regime, can be studied [10]. Driving forces may be both stochastic events (for example, violent storms, massive fish death, and herbicide use) and gradual processes that slowly degrade system resilience (eutrophication or global warming) [10]. The fact that a shallow lake is in a clear or murky regime implies that it presents distinctive feedback mechanisms as well as community structure and functioning. Thus, lakes in a clear water regime colonized by submerged macrophytes with a moderate–low plant coverage, have good light penetration, low pelagic primary production, zooplankton of large body size, high diversity of invertebrates and fish, and a high ratio of piscivorous to planktivorous fish [11]. In these systems, the control of zooplankton over phytoplankton (top-downregulation) is favoured because the piscivorous fish reduce the pressure of predation on the Cladocera community, made up of species of large body size and greater capacity for grazing, and also macrophytes might offer refuge to large cladocerans against planktivores [12]. In contrast, in systems with a turbid regime, phytoplankton become dominant because these lakes have a high level of nutrients and there is weak regulation from above as zooplankton are composed of species with less grazing capacity. Furthermore, the piscivorous to planktivorous fish ratio is low [6]. The shift from a clear macrophyte-dominated regime to a turbid one implies a drastic change on the balance and the interaction between the benthic-littoral and pelagic environments, with impacts on the fluxes of organic matter and nutrients, as well as the composition of the different assemblages and communities [6]. For example, the loss of macrophytes implies a change in the composition of the Cladocera assemblage with a decrease in the proportion of littoral and benthic representatives.

The stability of the zooplankton community in shallow lakes is strongly linked to small changes in the surface sediment condition (e.g., bank of seeds and eggs, physical changes of sediment). These changes cannot be detected by short-term studies because they are visible over long-time scales (e.g., decades or centuries) but can potentially be revealed by high resolution paleolimnological studies [9].

Lake sediments are rich sources of paleoecological information derived from the functioning of the lake ecosystem as well as the surrounding watershed. Sedimentary archives have been used to address several issues such as acidification, eutrophication, and lake ontogeny [13,14]. Among the proxies preserved in the sediment, algal pigments have been shown to have the same periodicity as fish fluctuations, and the deposition rates of pigments derived from edible algae are increased by the grazing activity of large-bodied zooplankton [15]. Further, herbivory creates novel derivatives such as pheophorbide *a* [16], and can increase the overall concentration of pigments in the remaining undigested material [16]. Thus, both accumulation rates and concentrations of sedimentary pigments are connected to the dynamic of zooplankton populations [17].

Size-selective fish predation has an impact on the zooplankton community structure, leading to the dominance of small-bodied species. As zooplankton remains are deposited in lake sediments in a direct and frequent way in proportion to zooplankton abundance, microfossils can be used to infer planktivorous predation pressure or even reconstruct fish stocks [18,19]. Several indicators of fish predation have been recognized as changes in ephippia size; the length of cladoceran mandibules, carapaces, or mucrones; or the planktivory index((*Daphnia*)/(*Daphnia*+*Bosmina*)) [11,20,21]. For example, the size of *Daphnia* ephippium has been demonstrated to be directly related to the size of female-bearing

eggs [22], whereas the length of the mucrone with the total length of the individual is the case for *Bosmina huaronensis* Delauchaux 1978 [23].

The main objective of this paper was to establish the occurrence of changes in the size of subfossil Cladocera remains and in the concentration of pheopigments in order to infer changes in the planktivory and herbivory pressure during the last 250 years in a shallow lake located on the Pampa Plain(Argentina).In this study, we identified changes in a herbivory biomarker on the size of *B. huaronensis* (mucrone) and the replacement of *Daphnia* species by a small cladoceran (*Bosmina*), indicating a change in two main ecological processes, grazing and planktivory over the last 250 years. The change in predation and grazing was mainly triggered by eutrophication. To evaluate changes in the planktivory and herbivory regimes, we determined the abundance of *Daphnia* and *Bosmina* species, the size of *Daphnia* species ephippia and *Bosmina* mucrone, the abundance of fish scales, and the concentration of pheophorbide *a*.

#### **2. Materials and Methods**

#### *2.1. Study Area*

Lake Blanca Chica (36◦50 00.9" S; 60◦28 00.9" W) (Figure 1) is located in the Pampa Plain area (Central Argentina, South America), which constitutes one of the largest areas of wetlands of South America [24]. The lake is a shallow (1–2 m), turbid (Secchi Disc depth: 0.2–0.3 m, chlorophyll *a* concentration: 90–500 mg m−3), warm temperature, and a polymictic lake of alkaline water (pH: 8–9.8). Currently, it is in eutrophic status (Total Phosphorus: 0.3–1.2 ppm), lacking submerged vegetation. The fish community is dominated by the planktivorous *Odontesthes bonariensis*(Valenciennes, 1835) in open waters and by the small fish *Cheirodon interruptus* (Jenyns, 1842) in the littoral zone. The zooplankton are composed of small–medium sized Cladocera, Rotifera, and Copepoda species [25].

**Figure 1.** Location of Lake Blanca Chica in the the Argentinean Pampa Plain, South America.

#### *2.2. Core Chronology and Paleolimnological Analyses*

A core of 60 cm was collected at the deepest part of the lake using a vibracorer on October 2015. Sub-sampling (every 1cm interval) was carried out on the entire core. Results described in this study correspond to the top 30 cm of the core. Below this level, no pigments or ephippia were detected. The age–depth model was developed applying the constant flux/constant sedimentation (CFCS) model to the radionuclide activities of 137Cs, 210Pb, and 226Ra (detected at the Radiochronology Laboratory of the Laval University, Canada). Three known chronomarkers were used to constrain the 210Pb model: the137Cs maximum peak at 6–7 cm as a time marker of A.D. 1964/1965; the first occurrence of *Eucalyptus* pollen (exotic tree)in the sedimentary record as a result of an extensive forestation since 1880 in Argentina; and a lithology change corresponding to an extreme regional dry pulse registered across the Pampean Plain at the end of the Little Ice Age (estimated to be A.D. 1775 ± 10 years) and recognized in other lakes, for instance, Mar Chiquita, Melincué [26,27]. For further details on the age–depth model, see [28].

Herbivory was inferred in determining the concentration of the pheophorbide *a* as it represents a grazer biomarker of invertebrate herbivory [20]. This pigment was extracted with 5 ml of a 90:10 acetone/MilliQwater solution overnight in the dark at 4 ◦C, after flushing with nitrogen from around 1 g of wet sediment. Then, sediments were centrifuged at 3000 rpm for 10 minutes before detection using high pressure liquid chromatography with an Ultimate 3000 system (Thermo Scientific, Waltham). The elution program and the methodology for pigment identification and quantification followed previous protocols [29].

Changes in the planktivory regime were inferred considering several indicators of fish predation: the abundance and size of *Daphnia* ephippia, the relative abundance and mucrone size of *Bosmina*, and the planktivory index((*Daphnia*)/(*Daphnia* + *Bosmina*)) [11,21]. This index was calculated considering the relative contribution of ephippia from *Daphnia spinulata* Birabén 1917, *Daphnia obtusa* Kurz 1875, and *B. huaronensis*, as chitinous remains from *Daphnia* species were very scarce or not found in the sedimentary record. Values of the planktivory index closeto 1 indicate a low planktivorous pressure, but when the ratio decreases it represents a higher fish predation pressure. Chitinous remains, ephippia, and fish scales were retained through washing 2–8 cm3 of sediment gently through a 50 μm sieve. Ephippia and fish scales were identified and were counted under a stereomicroscope at 10–40× magnification, whereas *Bosmina* remains were enumerated at 100–200xmagnification using a Zeiss Primo Star microscope. *Bosmina* remains were analysed on approximately a quarter of the sample after heating (70–80 ◦C) in 10% KOH, a deflocculating agent, for 45 min [30]. The length of the mucrone was considered as the distance from its base to its extreme, and the length of ephippia as the dorsal length, excluding anterior and posterior appendages. A total of 576 ephippia from *D. spinulata*, 324 from *D. obtusa*, 754 from *Moina* sp., and 1078 mucrones of *B. Huaronensis* were measured. At least 10 ephippia of each *Daphnia* species, 30 of *Moina*, and 30–50 mucrones of *Bosmina* were measured to calculate the mean size per stratigraphic level. The contribution of cladoceran species to the ephippia assemblage was expressed as weighted relative abundance [19], that is, ephippial relative abundance was weighted by that sample's total ephippial abundance. Several keys were used for taxonomic identification [23,31–33].

#### *2.3. Data Analyses*

The time series of the different biological indicators was modelled using generalized additive models (GAMs) [34]. In all the cases, fitted GAMs were estimated using maximum likelihood-based smoothness selection procedures, in particular the restricted maximum likelihood (REML). A continuous time first-order autoregressive process (CAR(1)) was chosen to account for the correlation between residuals [35]. To identify periods of transition, we estimated simultaneous confidence intervals from the posterior distribution of the model (under an empirical Bayesian formulation), and the first derivative of the fitted trend [35]. Periods of significant change are identified as those time points where the simultaneous confidence interval on the first derivative bounded away from zero [35]. The estimation of the models and derivatives were performed using the *mgcv* and *gratia* packages. In the case of the size of ephippia or mucrones, GAMs were fitted to the time series of the mean size for each indicator. Fish scales abundance and chitinous remains of *B. huaronensis* data were provided by González Sagrarioand co-workers [28].

To summarize changes in the ephippial assemblage, correspondence analysis was performed (package *vegan*) [36] and the number of axes that best explained data variance were selected according to the Kaiser–Guttman criterion and the broken stick model [37]. For further details, see Figure S1.

All analyses were performed using R version 3.5.1 [38].

#### **3. Results**

#### *3.1. Herbivory Biomarker*

The fitted GAM to the time series of pheophorbide *a* concentration explained a high percentage of data deviance and showed an increasing trend since ca. 1930. In particular, two increasing transitional periods occurred: ca. 1931–1949 and ca. 1991–2009 (Figure 2; Table 1), which indicate an increase in the total herbivory in the system.

**Figure 2.** Generalized additive model (GAM) fitted to the time series of pheophorbide *a* (expressed as η Molesg OM-1)in the sedimentary archive of Lake Blanca Chica. (**a**) Observed values, GAM-based trend fitted and its simultaneous interval; (**b**) estimated first derivative of the GAM-fitted trend and the 95% simultaneous interval; (**c**) period of transition.

#### *3.2. Ephippial Assemblage*

This study represents the first record of the ephippia of *Daphnia spinulata*, *Daphnia obtusa*, *Bosmina Leiderobosmina huaronensis*, *Moina* sp. Baird 1850, *Ceriodaphnia* sp. Dana 1853, *Leydigia louisi* Jenkin 1934cf, *Pleuroxus* sp. Baird1843, and *Chydorus sphaericus* (O. F: Müller 1776) group for the shallow lakes from the Pampa Plain. The weighted relative abundance of each species and the trend detected along the sedimentary record of Lake Blanca Chica can be found in Figures S1 and S2. Additionally, a decrease in the total sum of ephippia occurred after ca. 1915–1920, mostly represented by the loss of littoral species (Figures S1 and S3).

#### *3.3. Fish Predation Indicators*

The ephippia relative abundance and mean size of *Daphnia* species showed contrasting patterns. The effect of the smooth term for ephippia abundance and mean size was significant in all cases, and the fitted GAMs explained 25–38%of time series deviance (Table 1). *D. spinulata* showed an increasing trend in the mean size of the ephippium along the entire time series. In contrast, the relative abundance of ephippia in the sedimentary record showed an increasing trend since ca. 1785–1815 until ca. 1900, and after that a decreasing trend, diminishing its relative contribution to the lake zooplankton towards the present time (Figures 3 and 4). Similarly, *D. obtusa* showed an increase in ephippia mean size ca.1950–1995 and its contribution to the ephippial assemblage decreased since ca. 1900 (Figures 3 and 5). No trend for *Moina* ephippia contribution or mean size was detected (Table 1). Indeed, both variables showed a high inter-decadal/inter-annual variation (Figure 3 and Figure S1).

The fitted GAM to *B. huaronensis* relative abundance (chitinous remains) explained a high percent of the data deviance of the time series (81.7%) (Table 1). The first derivative of the fitted trend and its simultaneous interval showed an increase in *Bosmina* contribution ca. 1850–1880; after which no further change occurred (Figure 6). In contrast, a decrease in its size occurred ca. 1915–1935, stabilizing in a smaller size after 1940 (Figures 3 and 6; Table 1). For descriptive statistics on mucrone size, see Table S1.

**Table 1.** Results from generalized additive models (GAMs) fitted to the temporal series of the different proxies from the sedimentary record of Lake Blanca Chica. The estimated degrees of freedom (*edf*), *F*-statistic (Gaussian distributions) or *chi*-statistic (scaled or gamma distributions, values denoted by \*), and *p*-values of the smooth term and the percent (%) of the deviance explained by the fitted model are shown. The significance level was set at *p* < 0.05; ns: non-significant results. All the fitted models were estimated using continuous time first-order autoregressive process (CAR(1)) and restricted maximum likelihood (REML)smoothness selection.


**Figure 3.** Size of the ephippia of *Daphnia* species and *Moina* sp. and the mucrone of *Bosmina huaronensis* in the sedimentary record of Lake Blanca Chica.

**Figure 4.** GAM fitted to the time series of *Daphnia spinulata* ephippia abundance (left pannel) and ephippia size (right pannel) in the sedimentary archive of Lake Blanca Chica. (**a**) Observed values, GAM-based trend fitted and its simultaneous interval; (**b**) estimated first derivative of the GAM fitted trend and the 95% simultaneous interval; (**c**) period of transition.

**Figure 5.** GAM fitted to the time series of *Daphnia obtuse* ephippia abundance (left pannel) and ephippia size (right pannel) in the sedimentary archive of Lake Blanca Chica. (**a**) Observed values, GAM-based trend fitted and its simultaneous interval; (**b**) estimated first derivative of the GAM fitted trend and the 95% simultaneous interval; (**c**) period of transition.

**Figure 6.** GAM fitted to the time series of *Bosmina huaronensis* abundance (chitinous remians) (left pannel) and mucrone size (right pannel)in the sedimentary archive of Lake Blanca Chica. (**a**) Observed values, GAM-based trend fitted and its simultaneous interval; (**b**) estimated first derivative of the GAM fitted trend and the 95% simultaneous interval; (**c**) period of transition.

Fish scale abundance, an indicator of fish abundance, showed an increasing trend since ca.1930 to 2000 according to the first derivative of the fitted GAM. The model explained a high percentage of data deviance (64.4%) (Table 1; Figure 7). In concordance, a decreasing trend in the planktivory index occurred along the time series (Table 1; Figure 8). The GAM fitted to the planktivory index explained 29.2% of the deviance, and although it showed a decreasing pattern, it can also be observed that most of the values lower than 0.5 occurred after 1900 (Figure 8).

**Figure 7.** GAM fitted to the time series of fish scale abundance in the sedimentary archive of Lake Blanca Chica. (**a**) Observed values, GAM-based trend fitted and its simultaneous interval; (**b**) estimated first derivative of the GAM fitted trend and the 95% simultaneous interval; (**c**) period of transition.

**Figure 8.** GAM fitted to the planktivory index (*Daphnia*/(*Daphnia* + *Bosmina*)) estimated from the ephippial assemblage in the sedimentary archive of Lake Blanca Chica. (**a**) Observed values, GAM-based trend fitted and its simultaneous interval; (**b**) estimated first derivative of the GAM fitted trend and the 95% simultaneous interval; (**c**) period of transition.

#### **4. Discussion**

Predation and grazing are the principal processes that control community composition and size structure in lake ecosystems [6]. We found major changes in planktivory and herbivory in Lake Blanca Chica over the last 250 years. The alteration of zooplankton composition and size structure, the increase in the contribution of fish scales, and the decrease of the planktivory index are indicative of a shift in the planktivory regime. On the other hand, the increase of a biomarker of invertebrate grazing supports a change in the herbivory regime. Thus, this study shows a switch in the grazing and predation pressure and, as a consequence, in the fluxes of the pelagic food web.

*Bosmina* and *Daphnia* species showed opposite population dynamics in relation to their abundance prior to and post ca. 1900.The cladoceran assemblage changed its composition (ca. 1880–1900), shifting from being rich in littoral, benthic, and pelagic species (including *Pleuroxus*, *Chydorus, Leydigia, Ceriodaphnia, Moina, B. huaronensis, D. spinulata*, and *D. obtusa*) to being dominated by *B. huaronensis*. This fact is supported by the high contribution of chitinous remains (~80%) (Figure 6) and the appearance of *Bosmina* ephippia in the lacustrine archive post ca. 1900 (Figure S1). Several mechanisms can explain the abrupt change that occurred in the zooplankton community. The switch in the chitinous remains and in the diatom assemblages in Lake Blanca Chica (ca. 1880–1900) has been associated with an increase of the lake water level that affected the physical structure of the lake [28]. This abrupt change was triggered by the increase in precipitation since ca. 1870/1880 [39] and involved a switch in the relationship between the littoral and pelagic habitat and the loss of the aquatic vegetation [28]. Pelagic cladocerans perform dial horizontal migrations to littoral areas during the daytime, finding

refuge on the edge or inside submerged macrophyte stands to escape from planktivorous fish that occur in open water. However, neolimnological studies showed that the role of macrophyetesas refuge areas in subtropical temperate shallow lakes from the Southern Hemisphere differs from cold temperate shallow lakes from the Northern Hemisphere [5,12,40]. These studies were performed on eutrophic shallow lakes, and demonstrated that the lack of refuge provision in the southern lakes was due to the rich and abundant assemblage of predacious macroinvertebrates and small littoral fish that also seek refuge in macrophyte stands [5,12,40]. Despite this, the trophic status of Blanca Chica was not eutrophic before 1920–1930, according to a C/N relationship of around 25 [28]. Fish and macroinvertebrate abundance is directly correlated with primary production and nutrient level [6]. Thus, the abundance of small fish and predacious macroinvertebrates in the macrophyte stands must have been low given the C/N ratio of ~25; therefore, aquatic vegetation could have provided refuge to zooplankton, counteracting planktivory. Indeed, *D. spinulata* was favoured until 1900, as shown by an increasing trend. The increase in fish predation, evidenced by low values of the planktivory index, and the loss of the vegetation from the system (ca. 1880–1900) might have exposed zooplankton to a higher predation risk, making large-bodied species such as *D. spinulata* and *D. obtusa* more vulnerable to fish predation than small-bodied species such as *Bosmina*. Altogether, the change in the physical structure of the lake and the predation increase might have facilitated the decline of *Daphnia* species, and would have probably favoured *Bosmina* dominance, releasing it from *Daphnia* competition in addition to the benefit of its smaller size. The top-down regulation and the competition release are the most plausible mechanisms that led to *Bosmina* dominance because no enhancement of primary production (bottom-up regulation) was recorded for the period of the shift. The same mechanisms have been proposed to explain the shift from *Daphnia* to *Bosmina* in other lakes [41].

The mucrone (*Bosmina*) and ephippial (*Daphnia*) size decreases serve as independent indicators of planktivorous fish predation [3,21,42,43]. A shift in the planktivory regime occurred in the lake ca. 1930, indicated by the increase of fish scales since 1930 until present day and by the decreasing trend of the planktivory index (Figures 7 and 8). In this case, the higher top-down control on the zooplankton impacted the size composition of *Bosmina*, which showed a reduction in its body size during 1915–1930 (Figures 3 and 6). An important consideration in paleolimnological studies about *Bosmina* morphology is to obtain a statistically valid estimate of size structure [43]. A minimum sample size of 35 remains per stratigraphic level was determined as necessary to detect a statistically significant variation in size [44]. In our study, we measured between 30–60 remains per sample of sediment; thus, the mucrone size reduction results detected are valid, accurate, and act as an independent indicator in supporting the shift in the planktivory regime and the enhanced top-down control. The main driver of this shift was the eutrophication process that the lake underwent between 1920 and 1990 [28]. Our findings are in agreement with several studies that have recorded changes in the zooplankton composition and size structure as a result of an eutrophication process, leading to an increase of pelagic taxa, a replacement of large-bodied *Daphnia* by small-bodied *Bosmina* or *Ceriodaphnia*, and/or changes in the body size of pelagic taxa in different types of lakes (deep and shallow) [41,45,46].

Planktivorous fish select the larger species or individuals; thus, zooplankton display different strategies such as the reduction of its body size to counteract fish predation [4]. The increase in *Daphnia* body size or the lack of a specific trend in *Moina* sp. constitute the opposite expected patterns under an increasing fish predation scenario; then, it cannot be associated to planktivory. Even though, it can be explained by other mechanisms. *D. spinulata* increased its size over the entire time series whereas *D. obtusa* did so since ca.1950, that is, during the eutrophication phase. Jeppesen et al. [21] found that for a given CPUE (fish catch per unit of effort), *Daphnia* ephippia size apparently increased with total phosphorous (P). All *Daphnia* species are sensitive to food quality, requiring algae rich in phosphorous (P) content [47], and small *Daphnia* species, such as *D. spinulata* or *D. obtusa*, are favoured in high P lakes as they are less adversely affected by cyanobacteria filaments than large *Daphnia* species [48]. Therefore, a possible explanation is that the ephippiasize is bottom-up regulated, as no invertebrate predator occurred in this lake (such as *Chaoborus* or other cladocerans) and thus an increase in ephippia

size could not be induced. In the case of *Moina*, a summer species in shallow Pampean lakes, the high inter-annual/decadal variation in abundance and size could be associated with a low selection pressure and/or a mismatch with young of the year fish during this period.

Considering the different responses found in this study and combining *Bosmina* and *Daphnia* abundance and size trends, it is clear that they cannot be explained by a specific type of control. The explanation most likely lies in the interaction of an increase in nutrient concentration in the system (rendering a higher zooplankton production and higher *Daphnia* size) and a top-down control by planktivorous fish (impacting on the composition of the pelagic assemblage and the size of the dominant pelagic cladoceran, *Bosmina*).

The pheophorbide *a*, a grazer biomarker of invertebrate herbivory, has increased over two periods in Lake Blanca Chica: ca. 1930–1950 and 1990 until the present day. The increasing trend and the detected transitions also coincide with the eutrophication process in the lake and with the increase of the total sum of chitinous remains (represented by pelagic, benthic, and littoral species) [28], implying an increase of the overall grazing pressure in the system. The production of this pigment results from a higher food availability (bottom-up regulation) and a top-down control of herbivorous invertebrates, even when in the cladoceran assemblage the large-bodied species were replaced by small-bodied species with less grazing capacity. It seems that the higher zooplankton production counteracts, at least in part, the loss of species with higher clearance rates.

In this study, we found shifts in the herbivory and the planktivory regime in Lake Blanca Chica over the last 250 years, with impacts on different trophic levels and also on different attributes of populations and communities. The shift in herbivory implied a higher control over lake primary producers and, probably, a major flux of pelagic, and also littoral/benthic, carbon to higher trophic levels. Changes in predation pressure led to compositional changes in the zooplankton, with the replacement of large-bodied *Daphnia* by small-bodied *Bosmina*, and also to a reduction of body size in the latter. Shifts in predation pressure were first (ca. 1880–1900) related to a natural driver (hydrology) and then reinforced by eutrophication (ca. 1920–1930). The first driver induced an increase in predation risk due to the loss of refuge in littoral areas and the second one by an increment in the lake fish stock. Thus, we demonstrated that human activities and climate change affect relevant lake processes such as grazing and predation, inducing profound impacts on the function, structure, as well as in fluxes across lake food webs. In addition, our findings represent the first record of the ephippial assemblage for Pampean shallow lakes, showing the baseline condition for these lakes. These results should be taken into consideration when managing restoration activities are carried out in these degraded shallow lakes. Lastly, we emphasize and agree with previous results [49], pointing out that cladocerans are a strong candidate for the single best indicator in paleoecological studies related to the alteration in food web structure and changes in trophic status in shallow lakes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4441/12/2/597/s1: Figure S1: Ephippial stratigraphy of Lake Blanca Chica, including the scores from the first and second axes of correspondence analysis. Figure S2: GAM fitted to the time series of scores of the second axis (CA2) of the correspondence analysis estimated for the ephippial assemblage in the sedimentary record of Lake Blanca Chica. Figure S3: GAM fitted to the time series of ephippia (total sum) recorded in the sedimentary archive of Lake Blanca Chica. Table S1: Descriptive statistics for the size of the ephippia (μm) of *Daphnia spinulata*, *Daphnia obtuse*, and *Moina* sp., as well as the mucrone of *Bosmina huaronensis* (μm).

**Author Contributions:** Conceptualization, M.d.l.Á.G.S.; methodology, D.C., F.E.C., and M.d.l.Á.G.S.; validation, M.d.l.Á.G.S.; formal analysis, D.C.; investigation, D.C., S.M., A.L., F.E.C., and M.d.l.Á.G.S.; resources, S.M., A.L., and M.d.l.Ã.G.S.; writing—original draft preparation, S.M., A.L., and M.d.l.Á.G.S.; writing—review and editing, D.C., S.M., A.L., F.E.C., and M.d.l.Á.G.S.; visualization, D.C.; supervision, M.d.l.Á.G.S.; project administration, A.L. and M.d.l.Á.G.S.; funding acquisition, A.L. and M.d.l.Á.G.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by National Council of Scientific and Technical Research CONICET (grant: PIP 465/2013), National Research Council (CNR), Water Research Institute (IRSA), Bilateral Cooperation Project CONICET-CNR/2015 and Mar del Plata University (EXA775/2017).

**Acknowledgments:** We thank A. Kotov for all the material that was shared to M.d.l.Ã.G.S., J.C. Paggi for his help with Cladocera identification, to the three anonymous reviewers that contributed with their comments to improve this manuscript, and to G. Free and R. Burks for the English style edition.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Evaluation of the Egg Bank of Two Small Himalayan Lakes**

#### **Roberta Piscia 1,\*, Sara Bovio 2, Marina Manca 1, Andrea Lami <sup>1</sup> and Piero Guilizzoni <sup>1</sup>**


Received: 18 December 2019; Accepted: 6 February 2020; Published: 11 February 2020

**Abstract:** High mountain lakes are biodiversity treasures. They host endemic taxa, adapted to live in extreme environments. Among adaptations, production of diapausing eggs allows for overcoming the cold season. These diapausing eggs can rest in the sediments, providing a biotic reservoir known as an egg bank. Here, we estimated changes in abundance of the egg bank in two lakes in the Khumbu Region of the Himalayas, during the last ca. 1100 and 500 years, respectively, by analyzing two sediment cores. We tested viability of the diapausing eggs extracted from different layers of the sediment cores under laboratory conditions. We found that only diapausing eggs of the Monogont rotifer *Hexarthra bulgarica nepalensis* were able to hatch, thus suggesting that a permanent egg bank is lacking for the other taxa of the lakes, not least for the two *Daphnia* species described from these sites. Our results confirm previous studies suggesting that in high mountain lakes, the production of diapausing is mainly devoted to seasonal recruitment, therefore leading to a nonpermanent egg bank. The different ability of different taxa to leave viable diapausing eggs in the sediments of high mountain lakes therefore poses serious constraints to capability of buffering risk of biodiversity loss in these extremely fragile environments.

**Keywords:** zooplankton; diapausing eggs; high mountain lakes; Himalayas

#### **1. Introduction**

High mountain lakes are highly vulnerable to impact of human activities and to climate change [1–3]. They host endemic populations of aquatic invertebrates, adapted to live under extreme environmental conditions using different mechanisms for optimizing life-cycles and growth during a very short ice-free period and under the ice cover [3,4].

Research on high mountain crustacean populations has mainly focused on physiological adaptation mechanisms, such as development of cuticular pigmentation in cladocerans (melanin) [5,6] and diet dependent carotenoid pigmentation, in copepods [7,8]. Less well documented are zooplankton reproductive traits and their importance as survival strategies through time. Life-cycles of high mountain zooplankton taxa are characterized by cyclical parthenogenesis, where a shift from parthenogenetic to sexual reproduction occurs at the end of the ice-free period leading to production of diapausing eggs (embryos at early developmental stages). These stages are capable of surviving the harsh seasons and under the ice cover, securing recruitment of individuals into the population during the ice-free period [9,10].

The production of diapausing eggs with delayed development (undergoing diapause or dormancy) can be regarded as a mechanism through which taxa maintain their genetic reservoirs—buffering the high risk of extinction to which their populations are exposed [11–15]. Evaluating the "egg bank" potential allows us to estimate the vulnerability of different taxa to extinction and the risk of biodiversity loss in these environments. The dormant egg bank can in fact influence the rate and the

direction of population, community, and ecosystem response to climate change [12,14,15]. Under global environmental changes, the frequency of extreme conditions will likely increase and species with prolonged diapause will be able to survive extreme years of no recruitment, whereas species lacking an egg bank will not. An altered environment may change species having poor recruitment and even which species produce an egg bank, through effects on thermal diapause cues [16].

Information on remote high mountains environments is usually scant, limited in time and space, and even a list of taxa from contemporary samples is generally incomplete as plurennial surveys can be rarely accomplished. To this aim of investigating spatial and temporal variability in biodiversity and time changes, integration of paleo- and neo-limnological approaches is very useful. Analyses of sediment cores allow for reconstructing long-term changes in biodiversity of biotic communities along with changes in the environment, allowing for reconstructing changes in climate and in different types of human impacts, such as long distance transport of pollutants.

In this study, we trace long-term changes in abundance of diapausing eggs in two small lakes, LCN10 and LCN70, located in the highest mountains of the world. Diapausing eggs were extracted from two short sediment cores representing different stages of the lake life history and through different climatic conditions. Abundance and viability of diapausing eggs in the sediments are crucial for complementing contemporary biodiversity estimates with potential biodiversity. Consistency of the active egg bank in the two lakes was estimated by hatching diapausing eggs recovered from the sediment cores under controlled laboratory conditions. The aim was to understand risk of loss of endemic species and vulnerability of different taxa to impact of perturbations in these fragile environments, in which impact of climate change and of anthropogenic activities is high.

Our study was conceived in the framework of an extensive multiannual study aimed at characterizing lakes in the Sagarmatha National Park, Nepal. Both water samples and sediment cores were collected during fifteen limnological surveys carried out in the period 1992–2011. Of the 120 cataloged lakes in the region, ca. 70 were characterized for hydrochemistry, 40 of which were analyzed for plankton and benthos, providing taxonomic and biodiversity estimates in different years [17]. Subfossil Cladocera remains also allowed for understanding changes in biodiversity during different climatic phases [18].

#### **2. Study Sites**

LCN10 and LCN70 are located in the upper Khumbu Valley (Table 1; Figure 1), in the Nepalese Himalayas [19,20]. They are small and shallow lakes, with an ice-covered period of 9–10 months and a high transparency during the open water phase. LCN10 receives water primarily from LCN9 located upstream and from snowfields, while LCN70, classified as a temporary lake by Löffler [21], receives water only from snowfields. Both sites are part of the 40 lakes in which plankton has been studied [22,23] in the framework of the Ev-K2-CNR project [24] developed in collaboration with the Nepal Academy of Science and Technology (NAST). Previous information on taxonomy, reporting endemic taxa, was provided by Löffler [21,25] as a result of an extensive sampling campaign in the Khumbu Valley. In previous studies, zooplankton samples were collected with plankton nets (126 μm mesh size in 1993 and 1994; 50 μm mesh size in 1997 and 2004) by vertical and horizontal hauls at the deepest point and from the littoral. Samples were only qualitative, as the amount of filtered water could not be estimated. The samples were preserved in buffered 10% formaldehyde and counted to obtain estimates of relative abundance of the different taxa. LCN10, the deep lake close to the Pyramid laboratory, was visited in September–October of each sampling campaign while LCN70 was visited only in October 1994 and 1997. The list of taxa of LCN10 includes *Arctodiaptomus jurisovitchi* Löffler, 1968 [26], *Daphnia dentifera* Forbes, 1893 [27], *Daphnia fusca* Gurney, 1906 [28], *Alona werestschagini* Sinev, 1999 [29,30] and among rotifers, *Ascomorpha* sp. LCN70 was characterized by the prevalence of the Monogonont rotifer *Hexarthra bulgarica nepalensis* [31], forming dense agglomerates and bearing diapausing eggs. In this lake, only the melanic *Daphnia fusca* was found along with the Anostracan *Branchinecta orientalis* Sars, 1901 [22,32–34]. A few *Euchlanis* sp. were also reported, along with bottom

dwelling *Ilyocriptus acutifrons* Sars, 1862 (Cladocera Mactrothricidae) and a few Harpaticoidae of the genus *Maraenobiotus* sp. [35]. However, given the restricted number of samples, the sampling method and the mesh size, the list of taxa from zooplankton samples is far from exhaustive. The Ev-K2-CNR Pyramid laboratory located in the region at 5050 m a.s.l. gave technical and logistical support for the investigations, allowing for sampling some lakes in different years between 1992 and 2011. Seasonal hydrochemistry analyses and experiments in lakes closer to the laboratory, a detailed description of the morphometry and the chemistry of lakes and the traits of their catchment basin is reported in Lami and Giussani [17]. Besides, paleolimnological and paleoclimatic reconstructions were investigated in LCN10 and LCN70 [36,37]. The region is characterized by a cold and dry climate with monthly precipitation lower than 200 mm. Monthly mean temperatures range between −7 ◦C (December) and 5 ◦C (June).

**Table 1.** Geographic coordinates and principal morphometric characteristics of the studied lakes.

**Figure 1.** A map of the Khumbu and Imja Khola catchment within the Sagarmatha National Park, Nepal in which are highlighted all lakes sampled in the framework of the EV-K2-CNR project since 1987 (G.A. Tartari, pers. comm.). Black triangles refer the mountain peaks; black squares refer to localities. Red squares highlight the study sites.

#### **3. Materials and Methods**

A short sediment core (04/1, 26 cm in length) was collected near the deepest zone of LCN10 in 2004 using a gravity corer (with 6 cm diameter Plexiglas tube). The age–depth model was established by correlation with a previous core 02/3, dated by 14C method. A detailed description is provided in Lami et al. [37]. About 200 years were represented in the topmost six-centimeter sections of the core (resulting in a sedimentation rate of 0.03 cm y<sup>−</sup>1), and the deepest section was dated as corresponding to ca. 900 CE. The topmost 5 cm were sliced into sections of 1 cm, while for the remaining core a section of 1 cm was taken every 5 cm. In total, nine subsamples were extracted from the core.

Sediment samples were stored in the dark at 4 ◦C until microscopic analysis, in order to preserve the material and stimulate subsequent hatching [38]. All the diapausing eggs' cases found in the sediment core sections were counted, including those which, being open, were empty. The latter may indicate changes in recruitment from diapausing eggs over time. Taxonomic identification was performed, comparing images of rotifer diapausing eggs cases, which are often species-specific [39], to photographic material from previous studies [23,40] and on the basis of a guide on rotifers' diapausing eggs [41], while Anostraca diapausing eggs was based on a study [35] and literature [42–44]. Diapausing eggs were extracted following the sugar flotation method [45] modified for rotifer diapausing eggs by Garcia-Roger [46]. Taxonomic identification, counting, measurements and sorting of ephippia and rotifer diapausing egg cases were performed using a Zeiss Stemi 2000-C microscope (Carl Zeiss™, Oberkochen, Germany), equipped with software for image analysis (Image-Pro Express 5.1 (Media Cybernetics Inc., Silver Spring, USA)). The presence of eggs in dark ephippia was verified only after viability experiments, as opening ephippia cases prior to hatching might have damaged the eggs. Extracted ephippia and rotifer diapausing egg cases were individually transferred into multiwell plates (with well oxygenated culture medium of 4 mL oligomineral water) and stored in a thermostatic cell at <sup>17</sup> ◦C, with light intensity of about 50 <sup>×</sup> <sup>10</sup><sup>3</sup> Lux and L:D photoperiod of 16:8 h, reproducing summer environmental conditions. Every 24 hours we recorded hatchlings and renewed the culture medium.

Ephippia and diapausing egg cases from LCN70 were extracted from a sediment core collected in October 1994 (core 94/2) with a gravity corer (of 6 cm diameter Plexiglas tube) in the deepest zone of the lake. The core, 11.5 cm in length, was cut longitudinally and sectioned into 2–1.5 cm slices. Chronology had been established by 210Pb; sedimentation rate was 0.1 cm y−<sup>1</sup> in the topmost 6 cm and 0.02 cm y−<sup>1</sup> between 6 and 12 cm [36].

No attempt was made to hatch cysts of the Anostracan *Branchinecta orientalis.*

Duration of incubation was 21 days. Eggs that did not hatch during this time period were considered as unviable.

#### **4. Results**

The analysis of Cladocera ephippia in core LCN10 04/1 sections revealed periods during which only one subgenus was present and periods during which both *Daphnia* (*Ctenodaphnia*) and *Daphnia* (*Daphnia*) coexisted. In particular, in sections 3–4 cm and 4–5 cm only *D.* (*Ctenodaphnia*) ephippia were present, while in the topmost section only *D.* (*Daphnia*) ephippia were found (Figure 2A). As expected from previous studies on this environment, the few ephippia found in the superficial section belonged exclusively to a *D.* (*Daphnia*). Contemporary zooplankton samples had identified the species as *Daphnia dentifera* [47]. *D.* (*Ctenodaphnia*) ephippia were of the melanic species of the region.

Apart from the deepest section, ephippia were much more abundant from 2 to 5 cm sections, while in sections 10–11, 1–2 and 0–1 cm, their abundance was low. The highest abundance of *D.* (*Ctenodaphnia*) ephippia (12,385 m<sup>−</sup>2) was found in the section 4–5 cm corresponding to ca. 1800 CE; a second peak was detected in the section 2–3 cm where both *D.* (*Ctenodaphnia*) and *D.* (*Daphnia*) ephippia were found (1415 ephippia m−<sup>2</sup> and 7431 ephippia m<sup>−</sup>2, respectively). The two *Daphnia* species co-occurred also in sections 10–11 and 1–2 cm. Ephippia were lacking from deeper sections (15–26 cm) of the core.

After 21 days of incubation, no hatchlings were recovered from the ephippia extracted from core LCN10 04/1. Opening and inspection of their content revealed that ephippia cases were either empty or with only one egg which we classified as nonviable.

**&25(/&1**

**Figure 2.** Abundance of ephippia in the analyzed sections of the sediment core 04/1 of LCN10 (**A**) and range of variation of their length (**B**). Vertical lines represent median values while horizontal whiskers represent the 95th percentile.

The two types of ephippia had different sizes (lengths), with those of *D.* (*Daphnia*) always smaller than those of *D.* (*Ctenodaphnia*). The latter decreased in size between the 3–5 and 2–3 cm sections and, in the latter section, co-occurred with those of *D.* (*Daphnia*). *D.* (*Daphnia*) ephippia at 2–3 cm section were in a relatively large range of sizes, along with smaller *D.* (*Ctenodaphnia*) ephippia size. Five ephippia in total were found in the 10–11 cm section, corresponding to ca. 1450 CE; two were from *D.* (*Daphnia*) and their length was 0.9 mm, corresponding to the largest value of the 2 cm section. The other three pertained to *D.* (*Ctenodaphnia*), and their range of size did not differ from those detected in upper sediment sections. The relatively small size of the only *D.* (*Ctenodaphnia*) ephippium in section 1–2 cm (1.35 mm) seems to confirm the trend towards decreasing size observed between deeper and more surficial sections of the sediment core. The decrease in size of *D.* (*Ctenodaphnia*) ephippia in recent times was statistically significant (F = 11.803, *p* < 0.001; Figure 2B). In particular, mean ephippia length was statistically different between sections 4–5 and 2–3 cm (*t* = 4.855, *p* < 0.001; Table 2) and between sections 3–4 and 2–3 cm (*t* = 2.467, *p* < 0.016), but the difference between sections 4–5 and 3–4 cm was nonsignificant (Table 2). Changes in size of *D.* (*Daphnia*) ephippia in different sections of the core were not statistically significant.

**Table 2.** Results of all pairwise multiple comparison procedures (Holm–Sidak method, overall significance level = 0.05) on size of *D.* (*Ctenodaphnia*) ephippia extracted from sediment core LCN10 04/1. Significant comparisons are highlighted in bold.


Microscopical analysis of qualitative planktonic samples taken in LCN70 in 1994 revealed that both crustaceans and rotifers were present. Rotifers dominated in terms of numbers, the most common taxon being *Hexarthra bulgarica nepalensis* in lakes of the region [25,31]. The sample was very rich in rotifer eggs (Figure 3), with young and ovigerous females forming dense agglomerates on detritus and filamentous green algae. Few *Euchlanis* sp. specimens were also found [35].

**Figure 3.** (**A**) *Hexarthra bulgarica nepalensis* from a plankton sample of LCN70 carrying a diapausing egg; (**B**) Hatching of a diapausing egg of *Hexarthra bulgarica nepalensis* recovered from sediment core LCN70 94/2; (**C**) Newborn of *Hexarthra bulgarica nepalensis* hatched from the diapausing egg; (**D**) SEM image of a *Brachionus* sp. open egg case recovered from sediment core LCN70 94/2.

The profile of core LCN70 94/2 (Figure 4) shows the presence of diapausing eggs of three Rotifera taxa in all the sections: the dominant *Hexarthra bulgarica nepalensis* and, in low abundance, *Brachionus* of two morphotypes as reported in Sudzuki [48] and Piscia et al. [49]. The latter two were not found in the zooplankton samples. *Hexarthra* diapausing eggs were abundant in all sections but tended to increase in deeper sections of the core, with a peak of 30.4 <sup>×</sup> <sup>10</sup><sup>6</sup> <sup>m</sup>−<sup>2</sup> at the 8–10 cm section. They were between 1% and 2.5% of the total resting eggs found between the 10–11.5 and 6 cm sections and represented 23% and 15% of the total abundance at the 2 and at 4–6 cm sections, respectively. Cladocera ephippia of *D.* (*Ctenodaphnia*) and of *Alona werestschagini*, gradually increased from bottom to top layers of the sediment core and were 4.5 times more abundant in the upper 2 cm section than in the deepest one (10–11.5 cm). Anostracan cysts were also found to vary in abundance (min <sup>=</sup> 1.8 <sup>×</sup> 105 cysts m<sup>−</sup>2; max = 5.1 <sup>×</sup> 10<sup>5</sup> cysts m−2). *Brachionus* resting egg cases were all open, therefore revealing that diapausing eggs produced might serve as seasonal recruitment of the species at the end of the ice cover phase. No attempt was made of hatching Anostracan cysts, as we had no indication of conditions for promoting their hatching.

Only *Hexarthra* diapausing eggs from the topmost 2 cm were still viable, with a high rate of hatching (63.6%). Given the estimated mean sedimentation rate of 0.06 cm y−<sup>1</sup> [36], diapausing eggs in this section were deposited in a timeframe of ca. 33 years before date of collection of the core. Individual hatching timing varied between 4 and 7 days.

**Figure 4.** Abundance of Cladocera ephippia and Anostraca cysts (**A**) and rotifer resting eggs (**B**) recovered from sediment core LCN70 94/2.

#### **5. Discussion and Conclusions**

The presence of diapausing eggs in different sections of the two sediment cores confirmed that their production is a fundamental survival strategy in high-altitude lakes. The accumulation in sediments of diapausing eggs of different species, generations and genotypes, represents a potential reserve of biodiversity [12,16], which buffers the consequent extinction risk from anthropogenic impacts and makes the population resilient to the settlement of invasive species [50,51]. Organisms able to survive in extreme environments, such as LCN10 and LCN70, belong to a small number of highly specialized species; nevertheless, the reservoir of diapausing eggs in the sediments was of the same order of size (103–105 eggs per m<sup>−</sup>2) of those reported from less remote environments [16].

Zooplankton can produce different types of diapausing eggs: from embryos at early developmental stages of monogonont rotifers, Branchiopods and Calanoid Copepods, to advanced larval stages (Cyclopoid Copepods) and finally to adults of Harparcticoids and Bdelloid rotifers [16]. In previous studies, microscopic analysis of contemporary (plankton) samples revealed that Rotifera, Cladocera and Anostraca produced diapausing eggs at the end of the ice-free period [23,35]. Anostracan cysts recovered from sediments of LCN70 were of the species *Branchinecta orientalis* previous found in LCN70 and LCN30 zooplankton samples, in which the presence of diapausing eggs was also reported [35]. Among cladocerans, we found ephippia of the endemic *Alona werestschagini* in LCN70, *D.* (*Daphnia*) ephippia in LCN10, and *D.* (*Ctenodaphnia*) ephippia in both LCN70 and LCN10. In LCN10, in particular, an alternation in the occurrence of *D.* (*Daphnia*) and *D.* (*Ctenodaphnia*) ephippia was conspicuous and a co-occurrence of the two in the transition periods was observed. In a previous investigation, the presence of melanic *D.* (*Ctenodaphnia*) and pale *D.* (*Daphnia*) was reported [52]. Ephippia of the former were dark because of cuticular pigmentation (melanic) while those of the latter were transparent. The two also differed in size and morphology; ephippia morphology is in fact a diagnostic trait for distinguishing between *Daphnia* of the *D.* (*Ctenodaphnia*) type and those of the *D.* (*Daphnia*) type. The former belong to an ancestral type clearly separated from the other during the phylogeny ca. 200 million years ago [53].

Ephippia isolated from the surface layers of the sediment core indicate the substitution in recent times of the subgenus *D.* (*Ctenodaphnia*) with the subgenus *D.* (*Daphnia*) in agreement with plankton samples previously analyzed [23]. As suggested by Nevalainen et al. [18], the presence of *D.* (*Ctenodaphnia*) ephippia in the sediment core of LCN10 could be related to phases of enhanced connectivity with LCN9, the source of a cascade system in which *D.* (*Ctenodaphnia*) is permanent. Such changes in connectivity may in turn, be related to glacial advance or retreat driven by climate change [36]. Ephippia size is positively correlated with *Daphnia* body size, larger females producing larger ephippia resulting in larger neonates [54,55]. Such correspondence is the basis for reconstructing changes in *Daphnia* body size with changes in the environment [56]. In fishless, high mountain lakes, a wide range of *Daphnia* size (reconstructed from fossils, such as postabdomens, head shields, mandibles and, of course, from ephippia [52,57]) is regarded as indicative of a longer growing season, i.e., a longer ice-free season, securing a larger number of molts during the parthenogenetic phase which precedes the appearance of males and ephippial females at the end of the growing season [58,59]. In a study on LCN40, the substitution of the melanic *D.* (*Ctenodaphnia*) by the pale *Daphnia dentifera* in the most recent period was interpreted as related to changes in climate, with an increase in precipitation resulting in increase of dissolved organic carbon. In the case of a sediment core with low accumulation rates, however, multiple seasons in variable numbers are integrated in a 1 cm section during different time periods. Results of hatching experiments indicate that ephippial eggs recovered from the sediments of both lakes were nonviable, thus suggesting that an active egg bank is lacking for this important component of the zooplankton population. The apparent lack of a permanent *Daphnia* egg bank has been reported also from other alpine lakes (e.g., in *D. umbra* from a lake in southern Norway [60] and from Lake Paione Inferiore in the central Alps [58,61]. Ephippia bearing only one egg (i.e., lacking the bilocular structure typical of *Daphnia* ephippia [58]) or having only one diapausing egg left, are often reported as nonviable, also in the case of lakes in temperate regions [62]. As suggested in the case of lakes in the Alps forming a cascade system, we might hypothesize that the Himalayan upper Lake Piramide Superiore might represent the biotic reservoir for *D.* (*Ctenodaphnia*) *fusca*, as suggested in previous studies [33].

*Daphnia* is an important member of pond and lake systems in the Himalayas and Tibet [63–66]. Most lakes of this region were sampled during the 1960s by Löffler [21], who reported the presence of melanic *Daphnia tibetana* Sars, 1903 [63], endemic to the region, and pale *Daphnia longispina* Müller, 1776 [67] (classified as var. *aspina* by Werestschagin [30] because of the lack of a carapace spine at the adult stage). The former swims during the day in bottom waters and the latter apparently is restricted to lakes with limited transparency. Such distinction between transparent, "*D.* (*Ctenodaphnia*) lakes" and turbid "*D.* (*Daphnia*) lakes" was not confirmed by multiannual studies and by sediment core analyses. Our results on ephippia abundance in core 04/1 of LCN10 therefore represent a further evidence of co-occurrence of the two *Daphnia* species in lakes of the region. Co-occurrence of the large, melanic *D.* (*Ctenodaphnia*) and the smaller, transparent *Daphnia dentifera* in LCN10, is likely enhanced by different habitats: while the former is usually found swimming near the littoral, the latter tends to swim in deeper water, minimizing effects of UV damage. Our observation confirms what was reported by Löffler [21], who found the melanic *Daphnia* swimming in the littoral and the pale one swimming in deeper lakes and/or in turbid ones [68]. Largely differing in size, the two species might be regarded as able to feed on different food particles. In a very interesting and pioneer study, however, Geller and Müller [69] demonstrated that, while the distances between the setulae of filtering combs increase with individual body length in different Cladocera taxa, they do not increase with body length in *Daphnia magna*, a *D.* (*Ctenodaphnia*) species. Sharing the same food particle size, therefore, might result in a strong competition between the two *Daphnia* species; literature suggests that the larger one would be favored, being able to grow and reproduce at lower food concentrations [70]. Therefore, the fact that the two tend to live in different habitats appears as the most reasonable explanation.

Since the first studies carried out during the Yale North India expedition [66] of 1932–1933, endemic populations were reported from lakes in the Khumbu Region of the Himalayas, particularly among the zooplankton: *Arctodiaptomus jurisovitchi* [19] and *Daphnia tibetana*. Among monogonont rotifers, *Hexarthra bulgarica nepalensis* [31] were described. *Hexarthra bulgarica* has been reported from several high altitude lakes: the Rila Mountains, Bulgaria [71], the Italian Alps [72,73], the Himalayas [74], the Khumbu area (East Nepal) in lakes within the subnival belt [21,25], the Rocky Mountains (Grouse Lake, Alberta, Canada) [31], the Andes [75–77] and the Sierra Nevada [78].

The taxonomy of the melanic *D.* (*Ctenodaphnia*) from this region is problematical, largely because incomplete descriptions have been published to date. Originally, three species were identified: *Daphnia pamirensis* Rylov, 1930 [64], *Daphnia fusca* and *Daphnia tibetana* (first described as *Daphniopsis tibetana*). These species have been synonymized in every pairwise combination, or all three merged in one species, over the last century [65,79]. In his *Daphnia* taxonomic revision, Benzie [80] synonymized the genus *Daphniopsis* with the *genus Daphnia*, and *D. pamirensis* with *D. fusca*. He also synonymized *Daphnia* specimens from the Khumbu Region in Nepalese Himalayas, referred to as *D. tibetana* by Löffler [21] and as *D. himalaya* by Manca et al. [33]. More recently, a detailed analysis and taxonomic revision of diagnostic traits of the four *D.* (*Ctenodaphnia*) species (*D. tibetana*, *D. fusca*, *D. pamirensis* and *D. himalaya*) from their original sites (Kyrgyzstan, Gokyo and Khumbu, Nepal), revealed that morphology of populations of *D. fusca*, *D. pamirensis* and *D. himalaya* did not substantially differ from each other [47]. Results on morphology were also confirmed by genetic analyses (based on three mitochondrial genes, 12S, 16S COI). According to the latter, Himalayan and Tian Shan populations are conspecific, while *D. tibetana* clearly differs from the other two. The latter belongs to a wider group of *D.* (*Ctenodaphnia*) species that include *D. atkinsoni* Baird, 1859 [81] and *D. mediterranea* Alonso, 1985 [82] complexes as well as *D. fusca*. While *D. tibetana* and *D. fusca* are two valid, well differentiated but distantly related species, *D. pamirensis* and *D. himalaya* are younger synonyms of *D. fusca*. Populations of *D. fusca* so far occur only in high mountain glacial lakes, while *D. tibetana* inhabits saline lakes of Central Asian mountain ranges and high altitude plateau. Molecular systematics of the non-pigmented *D.* gr. *longispina*, presenting phylogenetic analyses for individuals collected from the Himalaya (Nepal) and the Pamir (Tajikistan) mountain ranges [47] recently suggested that populations from Nepalese Himalayas actually constitute a lineage within the *D. dentifera* clade. *Daphnia* from Lake Rangkul (Tajikistan) pertained to *D. longispina* clade. Results suggest that the two morphotypes represented two different species, among the most widespread ones in the *D. longispina* complex. Each species was confirmed genetically in at least two biogeographic regions (Palearctic and Holarctic in case of *D. dentifera*; [83] and Palearctic and Ethiopian in case of *D. longispina* [84]). According to these results, distribution of both species is therefore extended: the Himalayan LCN10 represents not only the westernmost, but also the highest locality reported for *D. dentifera*. Similarly, Lake Rangkul is the southeastern most and also the highest known locality for *D. longispina*, as evidenced by molecular data. A recent study on *Daphnia* of the Tibetan Plateau based on molecular analysis (COI gene) revealed a higher species richness with respect to those described in the past, however six of the taxonomic units identified by DNA analysis agreed very well to classical taxonomy descriptions [85].

In LCN70, Rotifera dormancy does not seem to persist over multiple seasons, except for the species *Hexarthra bulgarica nepalensis*. The relatively high numbers of *Hexarthra* diapausing eggs' cases may be indicative of a high production of diapausing eggs is this lake. In rotifers, the ability to produce diapausing eggs varies from species to species, and not necessarily with environmental conditions [86]. Differently from most Monogononta, *Hexarthra* stem females can in turn be mictic in temporary ponds from Texas [87]. Mictic females tend to appear very early in the season in lakes of the Sierra Nevada [78] and in temporary ponds of the Chihuahuan desert [87]. Fertilized eggs in dried sediment probably regularly hatch in large numbers soon after flooding in LCN70. They hatch during a short period that marks the beginning of population growth. The timing of this hatching period could be controlled by temperature at the sediment surface, by physical factors that promote resuspension of diapausing eggs into an oxygenated and illuminated water and by biotic factors [88–91].

Our results indicate that some of these diapausing eggs remain unhatched in their egg cases and preserve well in the sediments deposited at the bottom of LCN70, year after year, forming an active egg bank [92]. If the eggs are buried in the sediments, their ability to hatch probably depends on physical and biological processes that mix the sediments and bring them to the surface where they can experience hatching cues. Sediment mixing is more pronounced in shallow waters, and therefore, buried diapausing eggs should have a greater chance of hatching there [68]. In the Himalayan temporary lake studied, during the ice-free period, the water level is subject to wide fluctuations and the life cycle may start after the summer monsoon inundation provides a hatching environment with well-oxygenated water and light. Changes in the proportion of diapausing eggs in different sections of the core might therefore also reflect changes in environmental conditions related to the monsoon.

Viability tests revealed that 64% of *Hexarthra* diapausing eggs of the uppermost two centimeters of the sediment core were able to hatch. This result is in agreement with previous studies which reported a range of hatching success between 10% and 88% [51,93–95]. Given the low sedimentation rate of this lake, the deposition time of diapausing eggs spans over three decades, thus the large variation of hatching time we observed (between 4 and 7 days) may reflect differences in age of the eggs. Consistent with our results, viable zooplankton diapausing eggs are recorded in the upper 4–6 cm of sediments [49,92,96–101]. Differences between environments can be related not only to differences in sedimentation rates but also to differences in the mortality rates of dormant stages, that can range between 1% y−<sup>1</sup> and 64% y−<sup>1</sup> [51,100,102,103]. In shallow ponds, as well as in our lakes, diapausing eggs are more exposed to stressing conditions (e.g., desiccation, UV radiation) than in deep lakes, and this exposure can accelerate the rate of degradation processes [100]. Nevertheless, there is evidence in literature of a high longevity, up to a maximum of 100 years [12,49,100,104,105].

*Brachionus* hatched resting eggs were found in the sediment core of LCN70. While reported from nearby lakes [22], *Brachionus* was not found in zooplankton samples of LCN70 at the time of sampling. It might have grown earlier in the season, thus leaving in the sediments the egg resting cases we found, although in lower numbers that those of *Hexarthra*. We expect that in these high mountain lakes, seasonal succession is similar to that already reported from similar systems in the Alps, in which small zooplankton (monogonont rotifers and immature stages of copepods) grows earlier in the season than large *Daphnia* [58]. The chance of finding taxa depends on time and space, and on the method used for sampling. Species rare at the time of sampling might be overlooked, and such might be the case for *Brachionus* in plankton samples of LCN70, which is shallow and with a rocky, heterogeneous bottom. Fossils from sediment samples generally complement taxa detection in zooplankton contemporary samples (see [18] for a list of Cladocera taxa found from the same sediment core from LPI that we used in the present study). Unfortunately, rotifers do not leave fossil remains in the sediments, and copepods are also very rarely reported, as they tend to quickly degrade. Therefore, the only chance for detecting their presence is by means of their diapausing stages. Production of diapausing eggs in copepods is rare and finding of viable diapausing eggs in lake sediments, namely of *Diaptomus sanguineus* Forbes, 1876 [106], as old as 350 years, is deserving of topmost journals [94]. On the contrary, production of diapausing eggs in monogonont rotifers, particularly *Brachionus*, forming an egg bank in the sediments is commonly reported. So, our inability to find unhatched eggs in the sediments appears contrasting with the standard very long diapause of most monogonont rotifers. Of course, it might be that, given rarity, these viable eggs were not found in our sediment samples.

In conclusion, despite producing diapausing eggs in non-negligible amounts, the Cladocera zooplankton taxa investigated in two Himalayan lakes do not seem leave a biotic reservoir in the sediments by means of viable diapausing eggs. *Daphnia* ephippia were in fact empty and/or with only one, nonviable egg. Similar results were obtained in previous studies, reporting that when a bilocular ephippium is partly empty, the remaining egg is usually nonviable. The presence of unilocular ephippium is also reported from high mountain lakes [58], and infertility of the ephippial egg inside is attributed to UV damage [107]. Therefore, in these high mountain lakes, the egg bank is restricted to the seasonal recruitment of active individuals into the population when the ice cover melts. The biotic reservoir of an active eggs bank seems restricted to one monogonont rotifer species, *Hexarthra bulgarica* *nepalensis*, whose diapausing eggs were successfully resurrected (hatched) in large numbers from relatively recent sediments.

We cannot, of course, exclude that we were truly unable to study them at best; we might simply think that, as suggested by Ruggiu et al. 1998: "Any information on freshwater organisms from a truly remote area could be intrinsically interesting" [108].

If our interpretation is true, we might be very concerned about the future of biodiversity of these lakes, so beautiful, so fragile. Given the low connections of these lakes with other aquatic environments, and their remoteness, our results highlight their high vulnerability to loss of biodiversity and of taxa which are unique and precious, super-exposed to impact of climate change and long distance transport of pollutants [109–111].

**Author Contributions:** Conceptualization, M.M.; investigation, M.M., R.P., S.B., A.L. and P.G.; formal analysis: R.P. and S.B.; writing—original draft preparation, M.M. and R.P.; writing—review and editing, M.M., R.P., A.L. and P.G.; visualization, funding acquisition, M.M. and A.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The present research was funded to M.M. by CNR RSTL (Basic research funds on special projects) n◦552 on "Evolution of biodiversity and evaluation of the egg bank of aquatic organisms of remote lakes". Sampling and basic sediment core analyses were supported by funds to A.L. from CNR, Research Project on "Impact of Global Change in high elevation remote sites".

**Acknowledgments:** We wish to thank Simona Musazzi for helping in sampling of the sediment core of LCN10 in 2004 and G.A. Tartari for sampling the core of LCN70 in 1994. We wish to express our gratitude to the two anonymous referees: their contribution was crucial for substantially improving a previous version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Zooplankton Size as a Factor Determining the Food Selectivity of Roach (***Rutilus Rutilus***) in Water Basin Outlets**

#### **Robert Czerniawski 1,2,\* and Tomasz Krepski 1,2**


Received: 21 May 2019; Accepted: 19 June 2019; Published: 19 June 2019

**Abstract:** Fish occurring in the outlets of water basins reduce the abundance of zooplankton. The study was performed at the outlet sections of the lake and waste stabilization pond of a sewage treatment plant. The aim of the study was to determine which zooplankton is chosen more often by the roach (*Rutilus rutilus*), those drifting from the waste stabilization pond or from the lake. The zooplankton from the pond was dominated by *Daphnia pulex* while zooplankton from the lake was dominated by small planktonic rotifers. We observed that the larger the plankter-victim's size, the faster the reduction of its number. The fish were more likely to feed on zooplankton drifting from the waste stabilization pond than from the lake. It was influenced by *D. pulex* individuals, attractive for fish due to their largest body size among the analyzed zooplankton. The significance of riverine zooplankton in the downstream food web may render this data even more important.

**Keywords:** *Daphnia pulex*; stream ecology; river dispersion; live organic matter; fish feeding

#### **1. Introduction**

Fish make the most significant contribution to reduction of zooplankton communities [1–5]. This is especially visible in lake outlets where drifting zooplankton constitutes a rich food base for fish [6,7]. Even though the phenomenon of cyprinids feeding on zooplankton in outlets of stagnant basins is very common, it remains poorly investigated and documented [6,7]. Flow-through lakes and reservoirs provide a rich source of zooplankton for river ecosystems because organisms are washed out of the upper sections of lakes into river outlets. There is scarce literature regarding the efficacy and food selectivity of fish feeding in outlets of stagnant water basins. Certain authors speculate that fish are the main factor reducing zooplankton biomass in outlets [6–8], while others prove the point by showing zooplankton in fish stomach contents [9]. However, there is a lack of studies showing the stomach content of fish in regard to the zooplankton food-base in a river and the size of zooplankton victims.

This can be verified by checking how fish in the outlets of water basins reduce the abundance of zooplankton ranging in size. However, in the natural environment, it is difficult to find lake outlets with a high amount of large plankters such as *Daphnia magna* or *D. pulex* because they are immediately eaten by fish in the lake or at the beginning of a lake outlet, and therefore cannot reach the lower sections of a river outflow from the lake [6,7]. Therefore, it was crucial to find reservoirs without

fish that allow large plankters to drift downstream in the outlet where their abundance is reduced by fish. One such reservoir is the retention pond of sewage treatment plants, in which biological purification of treated sewage takes place. This pond may abound with large planktonic crustaceans such as *D. pulex*, which reproduce freely if they do not encounter their greatest stressor, that is, fish [10]. Such conditions were observed in a sewage treatment plant in a small town. An abundant amount of zooplankton was found in the pond and its outlet, where *D. pulex* was identified as the quantitative dominant. Compared to other plankters, this species reaches a large size and therefore is probably more susceptible to fish predation. The greatest reductions were observed in the largest plankters, adult cladocerans, and adult copepods [5,9]. Zooplankton that drift from the waste stabilization pond find their way to the Drawa river, and mix with zooplankton drifting from Adamowo lake. A high quantity of zooplankton attracts a great cyprinid biomass to the outlet of Drawa, including the roach (*Rutilus rutilus*), which feeds on this zooplankton type. This is a good field of research to determine which zooplankton is chosen more often by the fish, either the one drifting from the waste stabilization pond or the one from the lake, the factors that characterize the fish (e.g., their size and age), and the factors that characterize the zooplankton (e.g., body size and abundance) and influence the fish's choice. Answering the above questions was the aim of the study. The significance of riverine zooplankton in the downstream food web may render this data even more important.

#### **2. Methods**

#### *2.1. Study Area*

The study was performed at the outlet sections of the waste stabilization pond in the town of Drawno and at the outlet of Adamowo lake (GPS: 53.2089568; 15.7502837; NW Poland) (Figure 1). The samples were collected at 5 sites: the Adamowo lake outlet into the Drawa river (RLO), the waste stabilization pond (POND), the outlet from the waste stabilization pond (PO), a site in the Drawa river 20 m below the pond outlet (R1), and a site in the Drawa river 40 m below the pond outlet (R2). There were 2 control samples from 2 sites, i.e., RLO and POND, as a source of dispersed zooplankton, with river current in other sites, i.e., PO, R1, and R2. The biological treatment of sewage, previously purified by physical and chemical methods and with the use of activated sludge, is carried out in the retention pond of the sewage treatment plant (POND). The pond is densely covered with submerged vegetation such as *Ceratophyllum demersum*. Great feeding conditions and no fish presence in the pond create the optimal conditions for the development of *D. pulex* [1–5,11,12]. Phytoplankton communities and chlorophyll *a* concentration were not determined during the study, although these findings could have been useful for determining feeding conditions for *D. pulex*. Chlorophyll *a* measurements, taken the following year during 4 water discharges, indicated that in the summer chlorophyll *a* concentration was 38–84 μg L<sup>−</sup>1. Consistent with other study results, the analysis of our findings indicated that the pond provided great feeding conditions for *D. pulex*. Several authors have indicated that there is a high positive correlation between chlorophyll *a* concentration and the density of *Daphnia* species [13,14].

The other sites (in river) were dominated by pelagic rotifers; such a pattern is common in lotic waters due to several reasons. Rotifers are smaller in size and thus may be avoided by fish [9]. Also, their sedimentation is reduced due to their smaller specific weight, and consequently the rotifers may drift long distances, carried by the river current [9,15]. Additionally, the short development cycle allows rotifers to reproduce successfully under favorable conditions even in running waters [16]. However, in the case of this study, the definite quantitative prevalence of rotifers is determined by the presence of a highly eutrophicated lake that provides favorable conditions for the development of zooplankton.

**Figure 1.** Scheme of the study area. RLO—river outflow from the lake; POND—waste stabilization pond of sewage treatment plant; PO—pond outflow; R1—site in the river, 20 m below the pond outflow; R2—site in the river, 40 m below the pond outflow.

#### *2.2. Sampling Methods*

The zooplankton samples were collected for 4 days when the biologically purified water was being discharged from the retention pond by the sewage treatment plant. The sewage treatment plant discharged water for 30 min daily. Zooplankton samples were collected at the above-mentioned sites 10 min before the water discharge was around midday.

At each site, 50 L of water was collected from the river current. The samples were collected using a Van Dorn 5-L water sampler (KC Denmark) at five depths: 20%, 40%, 60%, 80%, and at the surface. At each depth level, 10 L of water was collected to obtain 50 L of water. The water was filtered through a plankton net with a mesh of 30 mm. The samples were then concentrated to 150 mL and fixed in a 4–5% formalin solution. Five sub-samples (2 mL) from the stirred total sample were pipetted into a glass Sedgewick Rafter Counting Chamber. The samples were identified using a Nikon Eclipse 50i microscope. Similarly to methods applied in our research on the relationship between the reduction rate in drifting zooplankton abundance and the type of fish present in the pond, in the course of this study we divided zooplankton into 8 groups, according to the body size and environmental preferences [7]: (i) benthic rotifers (Bdelloidea, Colurellidae, *Mytilina* sp., *Euchlanis* sp., *Lecane* sp., *Cephalodella* sp.), (ii) planktonic rotifers (*Pompholyx* sp., Filinidae, Trichocercidae, Brachionidae, *Polyarthra* sp., *Synchaeta* sp.), (iii) *Asplanchna* sp., (iv) small cladocerans (Bosminidae, Chydoridae), (v) large cladocerans (Dapnidae and *Diaphanosoma* sp. except *D. pulex*), (vi) adult *D. pulex*, (vii) cyclopid nauplii, and (viii) copepods (adult copepods and copepodites). Because of their large size and their dominance in the pond and pond outlet, adult individuals of *D. pulex* were not included in the large cladoceran group, but instead were categorized as a separate group. Planktonic rotifers, benthic rotifers, and cyclopoid nauplii were considered the smallest plankters. *Asplanchna* sp., bosminids, and chydorids were considered medium-sized plankters, whereas Dapnidae (except for *D. pulex*), *Diaphanosoma* sp., and copepods were considered large plankters. The largest plankters observed were *D. pulex* individuals [17–19].

In the attempt to verify food selectivity of fish, 30 roach were caught from the pond outlet site (PO) on the same day when the zooplankton samples were collected. The fish were caught 5 min before the end of the water discharge from the waste stabilization pond by 3 anglers of the Polish Angling Association. The anglers cast fishing rods into the pond outlet, straight into the area where the fish gathered. The fish took the bait immediately. The number of fish needed for the study was caught in a range of 1 min 15 s to 2 min 45 s. None of the fish were discarded. The fish sampling was random, and the fish caught differed in size, mass, and age. We also intended to determine zooplankton selectivity by fish varying in size and age. Scales were used to estimate fish age. The fish were measured and weighed. Total length and mass values were used to calculate the fish condition factor. The means of biomasses and the total body length of fish are shown in Table 1.


**Table 1.** Mean values ± SD of total length, mass and condition factor of fish.

The anglers caught 3 fish species—the roach (*Rutilus rutilus*), the white bream (*Abramis bjoerkna*), and the bleak (*Alburnus alburnus*). However, since roach constituted most of the catches (96%) the study focused only on this species. Given the above, we could objectively determine fish foraging efficiency. Once caught, the fish were stunned and killed. Afterwards, fish digestive tracts were preserved in 4% concentration of formaldehyde and taken to the laboratory where their contents were identified.

Given the site locations, no special field sampling permits were needed to conduct our study. We confirm that the land owner (the Polish Angling Association) gave us the permission to conduct the study at the examined sites. All fish were caught in the presence of the fish lake owner. Therefore, no specific permissions were needed for these locations and activities, and no approval of the Institutional Animal Care and Use Committee (IACUC) or an equivalent animal ethics committee was required. The field studies did not involve endangered or protected species. All sampling procedures and experimental manipulations required for our study have been reviewed and specifically approved under the field permit.

#### *2.3. Statistical Analyses*

To check for significant differences in abundance between the zooplankton groups found at the sites, the Kruskal–Wallis test was used (*p* < 0.05). The same test was used to check for significant differences in zooplankton amount between all zooplankton groups found in fish stomachs (*p* < 0.05). To determine significant differences between sites in terms of abundance and zooplankton number in fish stomachs, we performed post-hoc multiple comparisons of mean ranks for all groups (*p* < 0.05). The correlations between fish age and amount of zooplankton in fish stomachs were checked using

Spearman's rank correlation (*p* < 0.05). The aforementioned test was used to calculate the correlations between zooplankton abundance at sites and the zooplankton amount in fish stomachs (*p* < 0.05).

We checked the model with the Poisson distribution for overdispersion and found that it did not fit the data (*p* < 0.05). Then we used a generalized linear mixed model with negative binomial error distribution and log link function. The measurement values were added as a random effect. Zooplankton group abundance in the pond (POND) and the lake outlet (RLO) were included as covariates. We hypothesized that species abundance in the pond should increase the abundance at the sampling sites in the river (PO, R1 and R2). For modelling we used the "Mass" package [20] in the R package [21] for the analyses.

#### **3. Results**

We observed each zooplankton group at all sites except for the PO (Figure 2). The greatest zooplankton density was observed on 20 August. On every day of the data collection, *D. pulex* were predominant in the pond and in the pond outlet (Figure 2). Other sampling sites were dominated by planktonic Rotifera. A significant reduction of this species below the lake outlet indicates that the fish can consume almost the total amount of *D. pulex* drifting from the pond (Figure 2, Table 2).

**Figure 2.** Abundance of zooplankton at examined sites.

The greatest and most significant reduction in the abundance of drifting zooplankton groups between the PO and the R2 was reported for *D. pulex* (*p* < 0.05) (Table 2). Such great reductions were not reported for other zooplankton groups drifting from the lake (*p* > 0.05) or found between the RLO and the R2 sites, which is a section longer than between the PO and the R2. Even though no significant differences were reported, we noted that the larger the plankter size, the greater the differences in zooplankton abundance between subsequent sites (Figure 2, Table 2).

The model with the Poisson distribution showed that the abundance of zooplankton in the RLO did not affect its abundance at other sites (*p* > 0.05) (Table 3). We found that abundance in the pond affected the abundance in other sites (*p* < 0.05) (Table 3). We found that sites R1 and R2 differed significantly from the PO site in terms of *D. pulex* abundance reduction (*p* < 0.05) (Table 3).


**Table 2.** Mean ± SD of the zooplankton groups abundance at examined sites (*n* = 4). Different letters in rows show significant differences between sites (post-hoc multiple comparisons of mean ranks for all groups *p* < 0.05).




**Table 3.** *Cont.*

Examination of roach stomach contents showed that their diet consists mainly of *D. pulex* (Figure 3) because it had the highest percentage contribution of the total plankters across all individuals (Table 4). Even though *D. pulex* dominated in the stomachs of fish of all ages, we observed that older fish consumed considerably more *D. pulex* (*p* < 0.05) than younger fish (Table 4).

**Figure 3.** Total number of zooplankton individuals in stomachs of total fish.

*Water* **2019**, *11*, 1281

A significant correlation between the fish age and the number of zooplankton group individuals in fish stomachs was observed only for *D. pulex* (*p* < 0.05) (Figure 4). Correlations between fish age and number of zooplankton individuals from other groups found in fish stomachs were insignificant (*p* > 0.05).

**Figure 4.** Significant Spearman correlations between fish age and abundance of *D. pulex* in fish stomach (*R* = 0.86, *p* < 0.0001).

We did not observe any relationship between the increase in abundance of zooplankton groups in the RLO or in the PO, and a significant increase in the number of individuals of these groups in fish stomachs (*p* > 0.05) (Table 5). The aforementioned correlation was significant only in the case of large cladocerans in the RLO.

**Table 4.** Mean values ± SD of percentage contribution of zooplankton abundance in fish stomachs. Different letters in rows show significant differences between fish-age samples (post-hoc multiple comparisons of mean ranks for all groups *p* < 0.05).




#### **4. Discussion**

The present study shows that the reduction in abundance of several large plankters in a short river section may vary from a few percent to almost 100%. Naturally, the larger the zooplankton individuals, the greater the reduction of their abundance [6,7,22]. It would seem that the body size of plankters was a key factor that affected the amount of reduction in species abundance [5,7,9,19,23]. Fish substantially reduced large invertebrate prey, i.e., *D. pulex.* This species dominates in fish stomachs, and therefore proves that *D. pulex* abundance has been reduced by fish. Several papers have indicated that fish have a significant impact on the reduction of drifting zooplankton abundance in rivers [6,7,9]. Our study showed that fish are a crucial factor that reduces the abundance of drifting zooplankton in rivers, and their reduction efficiency increases with zooplankton size primarily because preying fish prefer large-bodied zooplankton.

Fish of all ages presented with high condition values due to the consumption of large amounts of *D. pulex*, which constituted an easily accessible food source. Low standard deviation scores for the fish condition factor indicate small differences in this parameter. This means that all the fish had equal and unlimited access to *D. pulex* as a food base.

Examination of fish stomach contents showed that older fish consumed less small planktonic organisms and more large-bodied ones, so these findings were not surprising [24]. However, given the results of the present study, it is difficult to prove the same for organisms other than *D. pulex*, because this species dominated in the stomach content of all fish of all ages. For this reason, when analyzing the plankter abundance reduction it is worth focusing on that specific species.

It is worth considering the correlations between fish age and number of zooplankton group individuals in fish stomachs, which was significant only in the case of *D. pulex*. Perhaps the correlation would have also been significant in the case of relatively large plankters such as large cladocerans or copepods. However, the fish had a large density of large *D. pulex* individuals to choose from, and clearly selected for this species. Hence, a conclusion can be drawn that fish made clear food selectivity decisions amongst the biggest plankters, which confirms the assumptions and results of other authors [5,7,9,25]. Even though other large planktonic crustaceans were available, the fish preferred *D. pulex*. We observed a significant reduction in Copepoda only in the further river sections, which means that fish consume other larger plankters once *D. pulex* is no longer available.

The results of the present work also show that the higher the abundance of zooplankton groups in a lake outlet or in a pond outlet, the higher the number of these groups observed in fish stomachs. However, there are no significant correlations between these variables to unambiguously support this statement. Perhaps, had the *D. pulex* been unavailable, the fish would have consumed other zooplankton groups and consequently such a correlation would have been significant. Nonetheless, the fish avoided feeding on small plankters because they provide little energetic gain [9]. The results of our present study also confirmed this hypothesis.

The abundance increase of *D. pulex* in a pond outlet does not affect the number of individuals in fish stomachs. This means that each day the abundance of *D. pulex* in the PO was sufficient for the fish biomass who almost entirely reduced the abundance of *D. pulex* in the river each day. Gliwicz [1] documented a case of continuous zooplankton consumption by fish in Cahora Bassa, Africa, and found that a great amount of easily accessible large zooplankton biomass was consumed by fish even though their stomachs were full. According to Gliwicz [1], it was a natural reaction for fish who had easy and short access to energetic gain. Therefore, despite having a full stomach, fish consume as much food as possible. Presumably, fish in the pond outlet consumed all the available large zooplankton biomass. It also confirms the foraging efficiency of fish examined in our experiment. The results of *D. pulex* abundance for the examined sites show that fish can consume almost the total amount of this species. This means that the pond outlet has a small effect on the movement of significant *D. pulex* biomass into the river, and thus on the entry of organic nutrients into it. Another factor is the incorporation of nutrients, eaten by zooplankton, into the fish tissues, and which eventually take part in the circulation of biogenic compounds [24].

The presented work shows the importance in limiting the abundance of drifting zooplankton in rivers by fish and the role of the plankter body size. The larger the plankter-victim's size, the faster the reduction of its number. In future research, it would be worth focusing on the issue of the efficiency and feeding potential of one fish to determine what size of fish biomass can eat the entire biomass of zooplankton carried away from the pond. This is an important problem, because the analysis of the results of this work does not indicate whether the zooplankton was continuously eaten by the same fish, or if the already fed fish were replaced by hungry fish. Finally, it can be concluded that the fish were more likely to feed on zooplankton drifting from the waste stabilization pond than from the lake. This was influenced by *D. pulex* individuals, attractive for fish due to them having the largest body size among the analyzed zooplankton, irrespective of the *D. pulex* percentage in the total abundance of zooplankton. Moreover, zooplankton are a valuable source of nutrients [26].

**Author Contributions:** Conceptualization, R.C.; methodology, R.C. and T.K.; software, R.C. and T.K.; validation, R.C. and T.K.; formal analysis, R.C. and T.K.; investigation, R.C. and T.K.; resources, R.C. and T.K.; data curation, R.C. and T.K.; writing—original draft preparation, R.C.; writing—review and editing, R.C. and T.K.; visualization, R.C. and T.K.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
