Experimental Evolution Shows Body Size Decrease in Response to Hypoxia, with a Complex Effect on Plastic Size Response to Temperature.

AbstractThere is a scientific debate whether oxygen concentration may be a factor driving the pattern of size decrease at higher temperature. Central to this debate is the fact that oxygen availability relative to demand for living organisms decreases with increasing temperature. We examined whether rotifers Lecane inermis exposed to hypoxic conditions would evolve smaller sizes than rotifers exposed to normoxic conditions, using experimental evolution with the same fluctuating temperature but differentiated by three regimes of oxygen availability: normoxia, hypoxia throughout the whole thermal range, and hypoxia only at the highest temperature. Immediately after the six-month experiment (more than 90 generations), we tested the plasticity of size responses to temperature in three post-evolution groups, and we related these responses to fitness. The results show that normoxic rotifers had evolved significantly larger sizes than two hypoxic rotifer groups, which were similar in size. All three groups displayed similar plastic body size reductions in response to warming over the range of temperatures they were exposed to during the period of experimental evolution, but they showed different and complex responses at two temperatures below this range. Any type of plastic response to different temperatures resulted in a similar fitness pattern across post-evolution groups. We conclude that (i) these rotifers showed a genetic basis for the pattern of size decrease following evolution under both temperature-dependent and temperature-independent hypoxia; and (ii) plastic body size responds consistently to temperatures that are within the thermal range that the rotifers experienced during their evolutionary history, but responses become more noisy at novel temperatures, suggesting the importance of evolutionary responses to reliable environmental cues.

Aerobic scope does matter in the temperature-size rule, but only under optimal conditions.

We united theoretical predictions of the factors responsible for the evolutionary significance of the temperature-size rule (TSR). We assumed that (i) the TSR is a response to temperature-dependent oxic conditions, (ii) body size decrease is a consequence of cell shrinkage in response to hypoxia, (iii) this response enables organisms to maintain a wide scope for aerobic performance, and (iv) it prevents a decrease in fitness. We examined three clones of the rotifer Lecane inermis exposed to three experimental regimes: mild hypoxia, severe hypoxia driven by too high of a temperature, and severe hypoxia driven by an inadequate oxygen concentration. We compared the following traits in normoxia- and hypoxia-exposed rotifers: nuclear size (a proxy for cell size), body size, specific dynamic action (SDA, a proxy of aerobic metabolism) and two fitness measures, the population growth rate and eggs/female ratio. The results showed that (i) under mildly hypoxic conditions, our causative reasoning was correct, except that one of the clones decreased in body size without a decrease in nuclear size, and (ii) in more stressful environments, rotifers exhibited clone- and condition-specific responses, which were equally successful in terms of fitness levels. Our results indicate the importance of the testing conditions. The important conclusions were that (i) a body size decrease at higher temperatures enabled the maintenance of a wide aerobic scope under clone-specific, thermally optimal conditions, and (ii) this response was not the only option to prevent fitness reduction under hypoxia.

Multivariate analysis of activated sludge community in full-scale wastewater treatment plants.

We investigated changes in protozoa and metazoa community in relation to process parameters in activated sludge from four wastewater treatment plants (WWTPs) throughout the period of 1 year. Principal component analysis (PCA) showed that activated sludge from investigated treatment plants had different dominating species representatives and community composition mainly depends on individual features of the treatment plants. Redundancy analysis (RDA) showed that the temperature in bioreactors was the most relevant factor explaining changes in the microorganism community, whereas reduction rate of chemical oxygen demand (COD), biological oxygen demand (BOD5), suspended solids (SS), and total nitrogen (TN) did not sufficiently explain the variation in protozoa and metazoan community composition. The results indicate that in stable working WWTP it is difficult to find a pronounced link between activated sludge species composition, process parameters, and plant configuration. Applied multivariate analysis can be a valuable tool for the exploration of the relations between community composition and WWTP process parameters.

What may a fussy creature reveal about body/cell size integration under stressful conditions?

There is a growing amount of empirical evidence on the important role of cell size in body size adjustment in ambient or changing conditions. Though the adaptive significance of their correspondence is well understood and demonstrated, the proximate mechanisms are still in a phase of speculation. We made interesting observations on body/cell size adjustment under stressful conditions during an experiment designed for another purpose. We found that the strength of the body/cell size match is condition-dependent. Specifically, it is stronger under more stressful conditions, and it changes depending on exposure to lower temperature vs. exposure to higher temperature. The question whether these observations are of limiting or adaptive character remains open; yet, according to our results, both versions are possible but may differ in response to stress caused by too low vs. too high temperatures. Our results suggest that testing the hypotheses on body/cell size match may be a promising study system for the recent scientific dispute on the evolutionary meaning of developmental noise as opposed to phenotypic plasticity.

Evaluation of the accuracy of two simple methods for microscopic activated sludge analysis.

Biological microscopic analysis is a popular method employed in wastewater treatment plants worldwide for evaluating activated sludge condition. However, many operators still have reservations regarding its reliability. In this study, we evaluated and compared two methods of microscopic sludge investigation: the sludge index (SI) and the Eikelboom-van Buijsen method (EB). We investigated 79 activated sludge samples from nine treatment plants located in southern Poland over a 1-year period. For each sample, sludge volume index values were calculated and compared with the results of evaluation made on the basis of microscopic analysis. Additionally, the effluent quality was analysed in 45 of 79 cases, including investigation of suspended solids, biochemical oxygen demand, chemical oxygen demand, total nitrogen and total phosphorous. The sign test and Wilcoxon matched pairs test showed that a significant difference existed between the two investigated methods. General conclusions from both methods do not provide reliable information concerning nitrogen and phosphorus removal. The EB method had a tendency to be more conservative in its general conclusions than the SI method. Both are highly reliable for estimating activated sludge quality and solid separation properties.

Interaction Between a Bacterivorous Ciliate Aspidisca cicada and a Rotifer Lecane inermis: Doozers and Fraggles in Aquatic Flocs.

Activated sludge is a semi-natural habitat composed of macroaggregates made by flocculating bacteria and inhabited by numerous protozoans and metazoans, creating a complicated interactome. The activated sludge resembles the biological formation of naturally occurring floc habitats, such as "marine snow." So far, these two types of habitat have been analyzed separately, despite their similarities. We examined the effect of a bacterivorous ciliate, Aspidisca cicada, on the quality of the macroaggregate ecosystem by estimating (i) the floc characteristics, (ii) the proliferation of other bacterivores (rotifers), and (iii) the chemical processes. We found that A. cicada (i) positively affected floc quality by creating flocs of larger size; (ii) promoted the population growth of the rotifer Lecane inermis, an important biological agent in activated sludge systems; and (iii) increased the efficiency of ammonia removal while at the same time improving the oxygen conditions. The effect of A. cicada was detectable long after its disappearance from the system. We therefore claim that A. cicada is a very specialized scavenger of flocs with a key role in floc ecosystem functioning. These results may be relevant to the ecology of any natural and engineered aggregates.

Foam-forming bacteria in activated sludge effectively reduced by rotifers in laboratory- and real-scale wastewater treatment plant experiments.

Lecane inermis rotifers were shown to diminish sludge bulking due to their ability to ingest the filamentous bacteria in activated sludge. To determine if rotifers are also able to control branched actinomycetes, we investigated three other Lecane species (Monogononta). In a week-long experiment, only Lecane tenuiseta significantly reduced the density of Microthrix parvicella and Type 0092 filaments, but in a 2-week experiment, actinomycetes were significantly reduced by most of the tested monogonont rotifers: L. inermis, Lecane decipiens and Lecane pyriformis. Rotifers L. inermis originating from the mass culture were artificially introduced into real-scale wastewater treatment plant (WWTP) in two series. The WWTP was monitored for 1 year. Rotifer inoculation resulted in diminishing of M. parvicella and actinomycete abundance. The experiments showed that different species of rotifers vary in their effectiveness at limiting various types of filamentous organisms. This is the first report demonstrating that one of the most troublesome bacteria, branched actinomycetes, which cause heavy foaming in bioreactors, can be controlled by rotifers. Knowledge of the consumers of filamentous bacteria that inhabit activated sludge could help WWTP operators overcome bulking and foaming through environmentally friendly methods.

The effect of three different predatory ciliate species on activated sludge microfauna.

Bacterivorous ciliates play important roles in the functioning of activated sludge by reducing dispersed bacteria and enhancing flocculation. There are, however, no data on the resistance of this functional group of microorganisms to predation. Our experiment was conducted with activated sludge subsamples subjected to artificially introduced three predatory ciliates species. The two predator species originating from activated sludge were Oxytricha sp. and Spathidium spathula. Dileptus margaritifer was a "foreign" predator species. The latter was introduced to compare its effect with the influence of predators naturally occurring in activated sludge on the ciliates community potentially adapted to certain predation strategies. Results showed that introduction of predatory ciliates into the activated sludge did not significantly change the total abundance of protozoa but rebuilt bacterivorous ciliate communities. Introduced predators significantly affected the most numerous ciliate species from the genera Epistylis and Cyclidium. In the presence of D. margaritifer, the abundance of sessile, colonial ciliates (Epistylis sp.) was significantly lower compared to the control treatment and to the treatments with the other predators. The activated sludge ciliate community was the most affected by the introduction of the "foreign" predator - D. margaritifer, a large ciliate armed with toxicysts.

'Optimal thermal range' in ectotherms: Defining criteria for tests of the temperature-size-rule.

Thermal performance curves for population growth rate r (a measure of fitness) were estimated over a wide range of temperature for three species: Coleps hirtus (Protista), Lecane inermis (Rotifera) and Aeolosoma hemprichi (Oligochaeta). We measured individual body size and examined if predictions for the temperature-size rule (TSR) were valid for different temperatures. All three organisms investigated follow the TSR, but only over a specific range between minimal and optimal temperatures, while maintenance at temperatures beyond this range showed the opposite pattern in these taxa. We consider minimal and optimal temperatures to be species-specific, and moreover delineate a physiological range outside of which an ectotherm is constrained against displaying size plasticity in response to temperature. This thermal range concept has important implications for general size-temperature studies. Furthermore, the concept of 'operating thermal conditions' may provide a new approach to (i) defining criteria required for investigating and interpreting temperature effects, and (ii) providing a novel interpretation for many cases in which species do not conform to the TSR.

The Temperature-Size Rule in Lecane inermis (Rotifera) is adaptive and driven by nuclei size adjustment to temperature and oxygen combinations.

The evolutionary implications of the Temperature-Size Rule (TSR) in ectotherms is debatable; it is uncertain whether size decrease with temperature increase is an adaptation or a non-adaptive by-product of some temperature-dependent processes. We tested whether (i) the size of the rotifer Lecane inermis affects fecundity in a way that depends on the combination of low or high temperature and oxygen content and (ii) the proximate mechanism underlying TSR in this species is associated with nuclei size adjustment (a proxy of cell size). Small-type and large-type rotifers were obtained by culturing at different temperatures prior to the experiment and then exposed to combinations of two temperature and two oxygen conditions. Fecundity was estimated and used as a measure of fitness. Nuclei and body sizes were measured to examine the response to both environmental factors tested. The results show the following for L. inermis. (i) Body size affects fecundity in response to both temperature and oxygen, supporting a hypothesis regarding the contribution of oxygen in TSR. (ii) Large individuals are generally more fecund than small ones; however, under a combination of high temperature and poor oxygen conditions, small individuals are more fecund than large ones, in accordance with a hypothesis of the adaptive significance of TSR. (iii) The body size response to temperature is realised by nuclei size adjustment. (iv) Nuclei size changes in response to temperature and oxygen conditions, in agreement with hypotheses on the cellular mechanism underlying TSR and on a contribution of oxygen availability in TSR. These results serve as empirical evidence for the adaptive significance of TSR and validation of the cellular mechanism for the observed response.

Effect of high levels of the rotifer Lecane inermis on the ciliate community in laboratory-scale sequencing batch bioreactors (SBRs).

Due to its ability to feed on filamentous bacteria, the rotifer Lecane inermis has already been recognized as a potential control agent of activated sludge bulking, which is usually caused by the excessive growth of filamentous microorganisms. However, their effectiveness depends, in part, on their abundance. We studied the influence of high densities of L. inermis on the protozoan community in activated sludge from a wastewater treatment plant (WWTP) in 4 laboratory-scale sequencing batch bioreactors (SBRs). Two treatments and two controls were subjected to nutrient removal system in process similar to that used in a WWTP. The experiment lasted 9 days and was repeated in 24-h cycles, including phases of agitation with feeding, aeration and agitation and sedimentation with decantation at the end of the cycle. In total, 32 taxa were identified, among which 25 were ciliated protozoa, 4 were amoebae, 2 were flagellates, and one was a nematode. Rotifers were then introduced to 2 bioreactors at a final concentration of 500ind.mL(-1), and the taxonomic composition and abundance of the activated sludge microfauna were assessed 2, 5 and 8 days thereafter. The mean density of ciliates on the first day of experiment was 12,610ind.mL(-1) and diminished to 4868±432ind.mL-±432ind.mL(-1) in the control and 5496±638ind.mL(-1) in the rotifer-treated group on the last day. Thus, even extremely high densities of artificially introduced rotifers did not negatively affect the protozoan community. On the contrary, the protozoan community was more diverse in the treatment group than in the control.

Tolerance to Ammonia of Thulinius ruffoi (Bertolani, 1981), a Tardigrade Isolated from a Sewage Treatment Plant.

The acute toxicity of ammonia on Thulinius ruffoi (Bertolani, 1981), a eutardigrade isolated from a small waste water treatment plant (WWTP) in Poland, was estimated. Our results show that no active individuals survived a 24 h exposure to solutions equal to or higher than 125 mg/L of total ammonia nitrogen (NH3-N + NH4 (+)-N), which, under the conditions in our experiment, was equivalent to 1.17 mg/L of un-ionised ammonia (NH3). The LC50 concentration of total ammonia nitrogen was equal to 52 mg/L (or 0.65 mg/L un-ionised ammonia). Given that the norms for the concentration of ammonia in treated waters leaving WWTPs are usually several times lower than the LC50 for T. ruffoi, this species does not seem to be a good bioindicator candidate for WWTPs. In this paper we also note that various ecotoxicological studies use different methodological approaches and we suggest that a more uniform methodology may aid interspecific comparisons of LC50 values.

Effect of the rotifer Lecane inermis, a potential sludge bulking control agent, on process parameters in a laboratory-scale SBR system.

The influence of a high density of rotifers, which is known to be able to control filamentous bacteria, on the parameters of an activated sludge process was examined in four professional laboratory batch reactors. These reactors allow the imitation of the work of a wastewater treatment plant with enhanced nutrient removal. The parameters, including oxygen concentration, pH and temperature, were constantly controlled. The experiment showed that Lecane rotifers are able to proliferate in cyclically anaerobic/anoxic and aerobic conditions and at dissolved oxygen concentrations as low as 1 mg/L. In 1 week, rotifer density increased fivefold, exceeding the value of 2,200 ind./mL. The grazing activity led to an improvement in settling properties. Extremely high numbers of rotifers did not affect the main parameters, chemical oxygen demand (COD), N-NH(4), N-NO(3), P-PO(4) and pH, during sewage treatment. Therefore, the use of rotifers as a tool to limit the growth of filamentous bacteria appears to be safe for the entire wastewater treatment process.

The toxicity of selected trace metals to Lecane inermis rotifers isolated from activated sludge.

The aim of the study was to assess the toxicity of a range of trace metals to the rotifer Lecane inermis, a species tested as a potential biological tool to control activated sludge bulking caused by overgrowth of filamentous bacteria in wastewater treatment plants. LC50 values (concentration lethal to 50 % of individuals, mg dm(-3)) were ranked in the following order: Cu < Al < Fe < Zn < Sn < Mn. L. inermis apparently is more sensitive to metals than other aquatic species widely used as model organisms in ecotoxicological testing, making it potentially useful for quick ecotoxicological tests.

Toxicity of ammonia nitrogen to ciliated protozoa Stentor coeruleus and Coleps hirtus isolated from activated sludge of wastewater treatment plants.

We assessed the toxicity of ammonia ions to Stentor coeruleus and Coleps hirtus (Protozoa) isolated from activated sludge taken from two municipal wastewater treatment plants in southern Poland. Stentor coeruleus is a rarely occurring species in activated sludge, unlike the widespread Coleps hirtus. The mean LC50 values (concentration causing 50 % mortality) calculated for the 24 h tests differed hugely between the tested species: 43.03 mg NH(4+) dm(-3) for Stentor coeruleus and 441.12 mg NH(4+) dm(-3) for Coleps hirtus. The ammonia ion concentration apparently is an important factor in the occurrence of these protozoan species in activated sludge.