The results of this study are based on a whole-lake experiment which was carried out in Halsjärvi, a small humic lake in southern Finland (Figure 1), in 2004–2007. The mixing manipulation was performed in 2005–2006, so besides after the manipulation period, data are also available from one antecedent and subsequent summer seasons. In addition, we gathered similar physical, chemical and biological data from a nearby lake (Valkea-Kotinen). This information is used as a reference for considering the ecosystem responses to the artificial mixing. Basic information about both the study lake and the reference is provided in Table 1.

Many results from our experiment have already been published, including a thermodynamic model [MyLake] application for ecological forecasting [18], physical and chemical responses to the manipulation [21], effects on mercury methylation [23] and methyl mercury concentrations in fish [22]. In this paper, we focus on the metabolic and food web responses of the manipulation. More information about the physical, chemical and biological characteristics of the reference lake, Valkea-Kotinen (Table 1 and Figure 1), including biogeochemical processes and long-term changes, are given in the following publications [28,29,30,31]. Both lakes have high dissolved organic carbon content due to high load of allochthonous humic substances, a characteristic feature for small lakes in the region [4,7,32]. Both lakes are influenced by similar meteorological drivers, because they are close to each other (distance is 4.5 km; for more information on the climate and deposition of the area) [33,34].

The mixing was achieved by using a modified commercial aeration equipment (MIXOX, Water-Eco Ltd., Kuopio, Finland) by means of an electrically-driven propeller positioned at 1.5 m depth below a raft anchored at the deepest point of the lake, and by pumping water from the metalimnion into the epilimnion (for more details of the experimental design and technical issues, see [18,21]). The artificial mixing started in the lake at the end of May 2005 and in that year mixing finished at the beginning of September, when autumnal turn-over usually starts. In 2006, mixing started at the beginning of June and finished at the beginning of September. Artificial mixing was intended to increase the depth of thermocline by 1 to 1.5 m (Figure 2 and Figure 3), a change which was selected to correspond to the projected increase in the mean temperature of the lake at the end of the 21st century due to climate warming.

The study lakes were sampled weekly in 2004–2006 and every two weeks in 2007. Samples for water chemistry and plankton biology were taken from the deepest point of each lake which is almost in the middle of both water bodies (Figure 1). Measurements from entire water columns for in situ profiles of water temperature (Tw) and dissolved oxygen (DO) concentrations were taken from the middle of the lakes by YSI 55 combined temperature-oxygen meter (Yellow Springs Instruments Inc., Yellow Springs, OH, USA). In both lakes a thermistor chain (Minilog data loggers) was also placed at the deepest point. The epilimnion, metalimnion and hypolimnion sampling depths were chosen each time based on Tw and DO profile measurements. Samples were collected with 0.3 m long (volume 2.4 L) Limnos tube samplers (except for zooplankton, see below), depending on the stratification and purpose of the samples (water chemistry vs. metabolic measurements). Each water layer was sampled from top to bottom, thus producing a composite sample. As a result, the collected water volumes varied a lot between different layers and sampling times, although the final subsamples for water chemistry (pH, alkalinity, conductivity, colour, TOC/DOC, TP, TN, PO₄-P, NO₃-N, NH₄-N, cations, and chlorophyll a and d) and biological measurements (densities and biomasses of organisms as well as primary production and respiration of plankton) were the same. During the homothermal periods an integrated sample from the whole water column was also taken for chemistry. In the reference lake samples for water chemistry and biology were taken from the entire water column [for zooplankton] or from different depths as a one meter composite samples, except that samples for metabolic measurements were taken from several narrow water layers from the surface down to the metalimnion or hypolimnion. However, in this article only the epilimnetic samples from the reference lake were analyzed for comparison purposes except in the case of zooplankton. It should be noted that the water-column of the reference lake is even more steeply stratified than in the experimental lake, and thus the depth of the photic zone is only approximately 1 m and below that depth anoxia commonly prevails in summer [28,31].

At the very beginning of the mixing treatment in 2005 the water column was completely overturned due to too high pumping power. Although the power was almost immediately reduced by changing the pumping direction from top down to bottom up, it took some weeks for thermal and chemical stratifications to re-form (for more details on the thermal chemical conditions, see [21]). Although artificial mixing induced some chemical responses in the lake [21], only the key results necessary for interpreting the food web responses are briefly mentioned in this context.

However, before any chemical responses caused by the mixing manipulation, we need to look at the chemical changes which originated already in 2004 due to the exceptionally wet summer season. During a period of seven weeks from 12 June precipitation was nearly 350 mm, which is the highest ever measured summer precipitation in 1963 when the measurements started in the area. As a consequence, surface and subsurface runoff dramatically increased and the groundwater table also reached its maximum level [35]. Because of increased runoff the lakes received high load of inorganic nutrients and organic matter from their catchment areas [28,31]. Therefore, epilimnetic DOC, colour, TP, TN, NO₃, NH₄ and Fe concentrations increased 2- to 4-fold until the middle of August compared to the early June [21]. As a result of the increase in colour and DOC, light penetration into the lake radically diminished. The following year when the artificial mixing enhanced the depth of the epilimnion, high colour and DOC concentrations were found in the lake. As a result of the greater mixing depth and enhanced light attenuation the euphotic zone then represented only 50% of the epilimnion. When colour values gradually decreased in the course of the two summer seasons with artificial mixing, light conditions simultaneously improved. However, a major change in photic conditions only appeared in 2007 after the mixing treatment was finished.

Analysis of chlorophyll a and bacteriochlorophyll d, indicators of phytoplankton and green sulphur bacteria were made by hot ethanol extraction and the role of different pigments was estimated based on the absorbance ratio method [24]. All chemical analyses were made according to the standard methods [28,31] at the laboratory of the Lammi Biological Station, University of Helsinki. Phytoplankton samples were preserved by acid Lugol’s solution and later counted by inverted microscope with 300× and 600× of magnification [36].

Primary production was determined using an acidification and bubbling modification of the ¹⁴C-method with a 24 h in situ incubation in light bottles [28]. Respiration of plankton was determined as the increase in dissolved inorganic carbon (DIC) in dark bottles during 24 h in situ incubation. DIC was determined with the acidification and bubbling method [37].

Zooplankton samples were taken with a 1-m-long 6.5 L tube sampler from the surface down to 5 m depth at 1 m intervals. Sample water was sieved through a 50 µm mesh plankton net before preservation of the zooplankton samples by formaldehyde solution (final concentration 10%). Zooplankton were counted and results calculated as densities (ind.·L⁻¹) [29,38].

The benthic macroinvertebrate samples were taken with a tube sampler of 64 cm² from three littoral (0–1 m) sampling points and by a tube sampler of 54 cm² from three profundal (6 m) sampling points. In both lakes the samples were taken in September 2004–2007. Five lifts per sampling point were pooled to make a single sample. The area of one sample was considered to be 318 cm² in the littoral and 270 cm² in the profundal zone. The samples were sieved through a 0.5 mm mesh net, animals were picked out and identified to major taxonomic groups and densities (ind.·m⁻²) and biomasses (g·m⁻², ww) were calculated [38].

Fish community structure was examined during 2004–2008 in both lakes using Nordic multi-mesh survey nets [39,40] with an effort of eight nets in both lakes each year. To examine possible responses of fish growth to the thermocline manipulation, the age of perch was determined and 1st and 2nd year growth back-calculated for the years 2002–2007 from opercular bones according to Monastyrsky’s procedure [41,42].

Randomized Intervention Analysis (RIA), Ref. [43,44] was used to test for statistical significance of changes that can be ascribed to the manipulation. The RIA method is designed specifically for paired-ecosystem experiments, in which one ecosystem is manipulated while the other serves as a reference. The method does not assume that the two lakes are identical, but rather that both lakes respond similarly to natural fluctuations. The method requires a pre-treatment monitoring period during which the inherent difference between the lakes is determined, and then a treatment monitoring period. The method tests the hypothesis that the difference between the lakes changed when the treatment was initiated. In this study, RIA was used to compare paired, chronologically-ordered samples for the four-year data record of phytoplankton and zooplankton (one year pre-treatment, two years treatment, one year post-treatment) from the manipulated (Halsjärvi) and reference (Valkea-Kotinen) lakes. The RIA was done separately for the pre- and post-treatment periods. For the pre-treatment period only values for the year 2004 were used. Calculations of RIA were conducted in SAS 9.4 (SAS Institute, Cary, NC, USA). For other statistical tests, parametric and nonparametric according to the data properties, and for production of graphs, SigmaStat12.5, SPSS, SYSTAT 13 and Real Statistic soft-wares were used.

Supplementary data are given in Tables S1–S7.

In summer 2005 bacteriochlorophyll d (BChl d) concentration, an indicator of green sulphur bacteria (GSB), collapsed in the hypolimnion of the study lake immediately after the onset of the mixing, and the concentration stayed low (<9.5 mg·m⁻³) in comparison to the other summer seasons (>25 mg·m⁻³, Figure 4). In 2006 the concentration was at the same level as that before and after the mixing experiment. The occurrence of GSB correlated negatively with hypolimnetic DO and SO₄ concentrations.

In the hypolimnion of the reference lake, no equivalent decline in BChl d concentration and absorbance ratio (665:654 nm) took place in summer 2005. According to RIA analyses, the mixing induced changes in BChl d concentrations were significant (p < 0.01) when the entire water column in the study lake was considered (Table 2).

In the study lake, in summer 2005 the biomass of diatoms contributed 27% of the phytoplankton biomass, which was twice that in 2004, and in July–August, in particular, clearly higher than in the post-treatment year (2.4-times higher) and in the reference lake (see also Figure 5 with data for June–September). According to RIA analyses, the response was significant (p < 0.01) when the pre-treatment year was considered (Table 2). The biomass of Asterionella ralfsii, Fragilaria sp. and Aulacoseira distans in particular increased. The biomass of non-flagellated green algae (data not shown) increased in synchrony with diatoms and in 2006 the biomass of both groups was still above that in the period before mixing. Monoraphidium dybowskii, a common non-flagellate green alga, had clearly higher biomass during the artificial mixing than before or after the treatment. Along with Diatomophyceae, the response of Cryptophyceae to mixing was statistically significant (RIA, p < 0.05). In 2004–2007 Gonyostomum semen (Raphidophyceae) constituted, on average, 17% of phytoplankton biomass while during the manipulation experiment its share dropped down to 4% in 2005 and to 9% in 2006. In the reference lake its contribution was higher, and was highest (on average 74%) in summer 2005. In the metalimnion and hypolimnion phytoplankton biomasses showed no response to the mixing.

Despite these changes in the share of different taxonomic groups of algae to the total phytoplankton biomass, no major changes in chlorophyll a concentrations (Figure 6) could be found (RIA for epilimnion of Halsjärvi and 0–1 m depth of Valkea-Kotinen, Table 2). The only indication that mixing influenced phytoplankton biomass in the study lake was the slightly increasing trend of chlorophyll a since 2004.

The differences in primary production among the years were significant (Kruskal-Wallis test, p < 0.05) only at the 0.5 m and 1 m depths. In the reference lake PP remained at the same level with no significant differences between the three summer seasons. During the summer seasons (June tomid September) the mean PP varied in Halsjärvi varied between 72–122 mg·C·m⁻²·d⁻¹ and in Valkea-Kotinen between 83–94 mg·C·m⁻²·d⁻¹ (Figure 7).

In Halsjärvi summer respiration of plankton (June to mid September) varied between 268 and 334 mg·C·m⁻²·d⁻¹, but the differences between the seasons were not significant. In the reference lake respiration was lower varying between 117 and 202 mg·C·m⁻²·d⁻¹, and in 2005 it was significantly lower (t-test, p < 0.05) than in 2004 and 2006.

For the two major taxonomic groups of zooplankton, rotifers and crustaceans, slight responses to the artificial mixing were recorded in the average densities in the entire water column. Rotifer densities were lower in 2005 and 2006 than before the treatment indicating a statistically significant response to the mixing (RIA, p < 0.05, Table 2, Figure 8 and Figure 9). No significant responses were detected in the main groups of crustaceans (Table 2). Cladoceran densities were somewhat higher during the mixing of the study lake (Figure 8) but due to wide within-year variation the differences were not significant. Among the cladocerans, non-significant responses were detected as the densities of Bosmina sp. and Daphnia sp. slightly decreased whereas the density of Ceriodaphnia sp. increased. Differences in copepods were also small between lakes as well as between years when mean densities of the whole water column were considered (Figure 8). However, in the hypolimnion of Halsjärvi the densities of cladocerans and calanoid copepods were distinctly (t-test, p < 0.05) higher especially during the first mixing summer season than during the summer before and after the treatment.

The summer average number of cladoceran taxa varied in the epilimnion between 5.6 and 6.8, being highest in 2007 and lowest in 2004, and that of rotifers varied between 16 and 20, being lowest in 2005 and highest in 2007. The lowest species richness was always found in the hypolimnion. In all three water layers the number of rotifer taxa gradually increased from 2005.

During the mixing in 2005 and 2006 the density of protozoans increased significantly in the hypolimnion (Figure 10), and to some extent also in the metalimnion and epilimnion, which might be connected to the decrease in Chaoborus larvae in the water column (Figure 10).

Among the littoral macroinvertebrates, Ephemeroptera, Chironomidae and Asellus aquaticus were numerically the most abundant taxonomic groups (Table 3). On average, the annual mean total densities of macroinvertebrates were distinctly lower in the study lake (500–3600 ind.·m⁻²) than in the reference lake (5100–11,300 ind.·m⁻²), mainly due to almost an order of magnitude higher densities of chironomid larvae in the reference lake (Table 3). The annual mean biomasses varied between 5.9–8.2 g·m⁻² (ww) and 9.1–15.9 g·m⁻² (ww), respectively.

During the artificial mixing in 2005–2006, the total density of littoral macroinvertebrates (Figure 11) of Halsjärvi was significantly higher than in the year before the mixing (Table 3). Only a few significant differences were recorded among taxonomic groups, (Ephemeroptera and Trichoptera). In some cases the lack of significance was at least partly because of wide within and between year variation. The highest littoral macroinvertebrate density in the reference lake occurred in 2006 and no significant between-year differences were found, except for chironomid larvae (Table 3). Total biomass of littoral macroinvertebrates did not show any response to the mixing, mainly due to the irregular occurrence of large Odonata nymphs.

In the profundal of the study lake, the mean total density of macroinvertebrates varied between 100–500 ind.·m⁻² and in the reference lake between 360–3100 ind.·m⁻². The mean biomasses were 0.3–1.5 g·m⁻² (ww) and 0.7–8.2 g·m⁻² (ww), respectively. Chaoboridae larvae were dominant in both lakes but in the study lake their density decreased sharply in 2005 and 2006 both in the water column (Figure 10) and at the bottom (Figure 11). In contrast, Chironomidae larvae appeared in the profundal samples in autumn 2005, after the first summer of mixing, and were present to the end of the study although in low numbers (Figure 11). Chironomids were also found occasionally in the profundal of the reference lake, and in 2007 their mean density reached a level of 1100 ind.·m⁻², probably due to complete mixing of water column in spring 2007 (Figure 3).

The fish community in the study lake consisted of European perch (Perca fluviatilis), ruffe, (Gymnocephalus cernuus), northern pike (Esox lucius), roach (Rutilus rutilus), bleak (Alburnus alburnus) and bream (Abramis brama) while in the reference lake only perch and pike were present. The mean annual gillnet catches from both lakes varied between 6–36 fish individuals and 200–600 g of fish per net with an increasing but not significant trend (Figure 12).

Roach was the dominant species in the study lake making up 66–79% of the numerical catch and 48–59% of the biomass catch of gillnets. The corresponding proportions for perch were 19–30% and 8–32%. Ruffe appeared in the catches of the manipulated lake during the second year of mixing in 2006. Perch dominated the gillnet catches of the reference lake.

The back-calculated length at age 1 values for perch prior to the mixing in 2002–2004 decreased from levels of 60–65 mm to 50–55 mm in both lakes with no statistically significant difference between the lakes (Figure 13). During the mixing in 2005 and in 2006, the first year growth of perch returned to 60–65 mm in the study lake but not in the control lake. The second year growth of perch was significantly higher in the control lake than in the study lake before the mixing (Figure 13). Since the mixing was started the second year growth in the study lake increased but still remained lower than in the control lake.

The boundary layers can be important microhabitats, which support phototrophic green sulphur bacteria (GSB) [24,49], methanotrophic bacteria [53], several protozoan species [54], and processes such as mercury methylation [23]. Besides anoxic conditions, other necessary requirements for GSB include hydrogen sulphide and solar energy which they need for photosynthesis. If any of those is missing, the conditions are not favourable for GSB [24]. Therefore, the unintended overturn just at the beginning of the manipulation experiment in 2005 explained their disappearance in that summer season. Although the mixing did not continue more than a couple of days, seemingly that was long enough for the community to die. In 2005, light availability was also rather poor because of high allochthonous organic matter load in 2004, and a consequent increase in light attenuation.

A similar recovery of the GSB community may take place each year because of the autumnal collapse of the community due to the overturn [24]. It seemed that the pigment concentration of the community could double in one week, which suggests that the peak concentration (~70 mg BChl d L⁻¹) can be reached within a few weeks after ice-out. In comparison to many other lakes in the region, BChl concentration in Halsjärvi was rather low [24]. Potential grazers of GSB and methane-oxidizing bacteria [54,55] include, in particular, ciliates and other organisms capable of living in poor oxygen conditions such as Chaoborus larvae, and to some extent also Cladocerans [47]. In the reference lake, GSB were also present and as in Halsjärvi, the water column was completely mixed in autumn and in spring 2007.

Among the phytoplankton, diatoms responded distinctly to artificial mixing and can be seen as the winners, which was not a big surprise. They are not common in sharply stratified humic lakes [56] but diatoms can be abundant in larger humic lakes if the thermocline lies deeper in the water column [50,57]. Thus, the results were in good agreement with previous observations and experiments showing that turbulence and strong enough mixing may allow high diatom cell density [58].

In turbulent conditions some flagellate algae may suffer due to several reasons. For instance, they may not be able to optimize their position in the water column relative to light [59] and nutrients as they can do in more stable conditions [60], and because of that their growth rate may decrease with further implications for their contribution to phytoplankton biomass. One such species was Gonyostomum semen, a large flagellate alga which has the capacity to exploit the whole water column in shallow humic lakes [60]. It seems that the shift in mixing condition somehow hampered this species. The share of Gonyostomum in the phytoplankton biomass was clearly higher in the reference lake [28] than in the study lake.

Another algal genus, Cryptomonas, which is commonly found in small humic lakes and exhibits distinct diurnal migration patterns [60,61], also responded to the mixing. The biomass increase during the experiment was not surprising, because cryptophytes are abundant in a wide range of aquatic environments [50], including small and large humic lakes with shallow and deep thermoclines. They are also known to be mixotrophs and some species heterotrophs, properties which support their survival in harsh and unpredictable conditions. Cryptophytes provide high quality food for zooplankton [62]. However, the observed changes in the phytoplankton community composition did not indicate any major changes in the metabolic traits between autotrophy and heterotrophy.

Primary production did not markedly respond to the mixing, which was in agreement with the observation that only minor changes in nutrient concentrations took place in 2005 and 2006 relative to the pre- and post-treatment years [21]. Either no response to lower PAR level in 2005 was found.

The increase in protozoan density was among the major food web impacts of mixing. The clearest change took place in the hypolimnion where their density substantially increased during the manipulation. That was in line with our earlier observations that many protists occur close to the boundary layer between the epilimnion and hypolimnion [54]. When the upper layer of the hypolimnion was mixed with the rest of the lake, protozoans also drifted to the upper water column and produced there a slightly higher density than before the treatment. At the same time, the density of Chaoborus larvae declined in the entire water column to around zero, which reduced the predation pressure on protozoans and larger zooplankton.

Due to stronger water circulation, grazing by larger zooplankton might not be as efficient in controlling ciliates as before and after the treatment. We have earlier shown that when crustacean zooplankton was removed from a humic lake, ciliate numbers rapidly increased in the epilimnion [63]. The changes in the species relations of dominant cladocerans were comparable to those detected in the TIMEX experiment [19].

The increase in the density of littoral zoobenthos, especially Asellus aquaticus, Ephemeroptera, Trichoptera and Chironomidae, suggests improved conditions for benthic fauna after the mixing was started. The decreased occurrence of Chaoborus larvae in the years of mixing may be a consequence of increased fish predation due to improved oxygen conditions. The appearance of chironomid larvae in the profundal of the study lake in 2005 was also an indication of improved DO conditions. The higher density of Chironomids in 2005 clearly indicated improved redox conditions in the upper sediment core relative to 2004, when Chironomids were absent.

Although the animal numbers were low, this was a remarkable change in the ecosystem which could have major impacts on the food web in long-term. These include, for example, the exploitation of methane-oxidizing bacteria (MOB) as an alternative carbon source for zooplankton and zoobenthos [64], and which may extend the food resources of fish [65,66].

As expected, no clear responses to thermocline manipulation were recorded in species relations or relative abundances of the fish community in Halsjärvi. The thermocline manipulation simply may not cause any fish community responses, or possible the study period was too short to detect any such responses in longer-lived organisms. Furthermore, most fish species in Halsjärvi (perch, ruffe, pike and roach) are core species of Finnish lakes characterized by wide environmental tolerances and wide distribution in all kind of lakes [67,68]. On the other hand, the appearance of ruffe in the gillnet catches in 2006 may be a consequence of mixing that resulted in increased oxygenated benthic habitat suitable for ruffe [69]. In addition, the observed increase in the early growth of perch might be one of the best indications that water column manipulation influenced the entire food web and the responses were cascading to the upper trophic levels. It must be underlined that the significant difference in the first year growth of perch between the two lakes was mainly due to the decreased early growth in the reference lake [30,70] rather than to increased growth in the experimental study lake. The slower second year growth of perch in the study lake before the manipulation compared to the control lake may be due to the food competition with roach [71,72]. The increase in second year growth of perch during the mixing suggests enhanced food availability and is comparable to other observations of positive second year growth responses of perch to environmental changes [73,74]. Increasing growth of fish may also contribute positively to the ecosystem services of lakes.

Another example of cascading effects from the microbial community up to fish was the decrease of mercury [Hg] concentration in perch [22] which was related to the mixing-induced responses both in the mercury methylation due to sulphate reducing bacteria [23] and in increased contribution of methane derived carbon in the food web due to enhanced activity of methane-oxidizing bacteria (MOB). The role of MOB was seen as more negative δ¹³C‰ values of perch in Halsjärvi during the mixing as compared to the years before and after, when no similar response was detected in the control lake [22].

Methyl-Hg (MeHg) production was determined (indirectly) by DO concentration at the layers with intense sulphate reduction [23], indicating that water column stratification is a prerequisite for MeHg production. Mixing caused a decline in net methylation, which resulted in an immediate decrease in Hg concentration in small perch [22]. Thus, our experiment demonstrated that in small lakes with an anoxic hypolimnion, MeHg production and bioaccumulation in the food web can be manipulated [22,23]. Correspondingly, climate induced changes in DO and heat content may affect MeHg production in lakes. Our results were in line with the TIMEX results [25], where oxycline depth significantly influenced hypolimnetic MeHg concentrations. However, according to that experiment thermocline depth, anoxic water volume, interface area of oxic-anoxic water, and sediment area in contact with anoxic water did not have a significant effect on MeHg concentrations. According to the authors [25] increased pelagic primary and secondary production might cause zooplankton and fish MeHg decreases via algal and growth dilution, which is a different interpretation than we have offered in our study. Irrespective of the mechanism, decreasing fish MeHg will improve the ecosystem services of small boreal lakes.