Evolution and development of Drosophila melanogaster under different thermal conditions affected cell sizes and sensitivity to paralyzing hypoxia.

Environmental gradients cause evolutionary and developmental changes in the cellular composition of organisms, but the physiological consequences of these effects are not well understood. Here, we studied experimental populations of Drosophila melanogaster that had evolved in one of three selective regimes: constant 16 °C, constant 25 °C, or intergenerational shifts between 16 °C and 25 °C. Genotypes from each population were reared at three developmental temperatures (16 °C, 20.5 °C, and 25 °C). As adults, we measured thorax length and cell sizes in the Malpighian tubules and wing epithelia of flies from each combination of evolutionary and developmental temperatures. We also exposed flies from these treatments to a short period of nearly complete oxygen deprivation to measure hypoxia tolerance. For genotypes from any selective regime, development at a higher temperature resulted in smaller flies with smaller cells, regardless of the tissue. At every developmental temperature, genotypes from the warm selective regime had smaller bodies and smaller wing cells but had larger tubule cells than did genotypes from the cold selective regime. Genotypes from the fluctuating selective regime were similar in size to those from the cold selective regime, but their cells of either tissue were the smallest among the three regimes. Evolutionary and developmental treatments interactively affected a fly's sensitivity to short-term paralyzing hypoxia. Genotypes from the cold selective regime were less sensitive to hypoxia after developing at a higher temperature. Genotypes from the other selective regimes were more sensitive to hypoxia after developing at a higher temperature. Our results show that thermal conditions can trigger evolutionary and developmental shifts in cell size, coupled with changes in body size and hypoxia tolerance. These patterns suggest links between the cellular composition of the body, levels of hypoxia within cells, and the energetic cost of tissue maintenance. However, the patterns can be only partially explained by existing theories about the role of cell size in tissue oxygenation and metabolic performance.

Heat tolerance in Drosophila melanogaster is influenced by oxygen conditions and mutations in cell size control pathways.

Understanding metabolic performance limitations is key to explaining the past, present and future of life. We investigated whether heat tolerance in actively flying Drosophila melanogaster is modified by individual differences in cell size and the amount of oxygen in the environment. We used two mutants with loss-of-function mutations in cell size control associated with the target of rapamycin (TOR)/insulin pathways, showing reduced (mutant rictorΔ2) or increased (mutant Mnt1) cell size in different body tissues compared to controls. Flies were exposed to a steady increase in temperature under normoxia and hypoxia until they collapsed. The upper critical temperature decreased in response to each mutation type as well as under hypoxia. Females, which have larger cells than males, had lower heat tolerance than males. Altogether, mutations in cell cycle control pathways, differences in cell size and differences in oxygen availability affected heat tolerance, but existing theories on the roles of cell size and tissue oxygenation in metabolic performance can only partially explain our results. A better understanding of how the cellular composition of the body affects metabolism may depend on the development of research models that help separate various interfering physiological parameters from the exclusive influence of cell size. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Oxygen and temperature affect cell sizes differently among tissues and between sexes of Drosophila melanogaster.

Spatio-temporal gradients in thermal and oxygen conditions trigger evolutionary and developmental responses in ectotherms' body size and cell size, which are commonly interpreted as adaptive. However, the evidence for cell-size responses is fragmentary, as cell size is typically assessed in single tissues. In a laboratory experiment, we raised genotypes of Drosophila melanogaster at all combinations of two temperatures (16 °C or 25 °C) and two oxygen levels (10% or 22%) and measured body size and the sizes of cells in different tissues. For each sex, we measured epidermal cells in a wing and a leg and ommatidial cells of an eye. For males, we also measured epithelial cells of a Malpighian tubule and muscle cells of a flight muscle. On average, females emerged at a larger body size than did males, having larger cells in all tissues. Flies of either sex emerged at a smaller body size when raised under warm or hypoxic conditions. Development at 25 °C resulted in smaller cells in most tissues. Development under hypoxia resulted in smaller cells in some tissues, especially among females. Altogether, our results show thermal and oxygen conditions trigger shifts in adult size, coupled with the systemic orchestration of cell sizes throughout the body of a fly. The nature of these patterns supports a model in which an ectotherm adjusts its life-history traits and cellular composition to prevent severe hypoxia at the cellular level. However, our results revealed some inconsistencies linked to sex, cell type, and environmental parameters, which suggest caution in translating information obtained for single type of cells to the organism as a whole.

Oxygen Dependence of Flight Performance in Ageing Drosophila melanogaster.

Similar to humans, insects lose their physical and physiological capacities with age, which makes them a convenient study system for human ageing. Although insects have an efficient oxygen-transport system, we know little about how their flight capacity changes with age and environmental oxygen conditions. We measured two types of locomotor performance in ageing Drosophila melanogaster flies: the frequency of wing beats and the capacity to climb vertical surfaces. Flight performance was measured under normoxia and hypoxia. As anticipated, ageing flies showed systematic deterioration of climbing performance, and low oxygen impeded flight performance. Against predictions, flight performance did not deteriorate with age, and younger and older flies showed similar levels of tolerance to low oxygen during flight. We suggest that among different insect locomotory activities, flight performance deteriorates slowly with age, which is surprising, given that insect flight is one of the most energy-demanding activities in animals. Apparently, the superior capacity of insects to rapidly deliver oxygen to flight muscles remains little altered by ageing, but we showed that insects can become oxygen limited in habitats with a poor oxygen supply (e.g., those at high elevations) during highly oxygen-demanding activities such as flight.

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.

Sexual Dimorphism in the Multielemental Stoichiometric Phenotypes and Stoichiometric Niches of Spiders.

Nutritional limitations may shape populations and communities of organisms. This phenomenon is often studied by treating populations and communities as pools of homogenous individuals with average nutritional optima and experiencing average constraints and trade-offs that influence their fitness in a standardized way. However, populations and communities consist of individuals belonging to different sexes, each with specific nutritional demands and limitations. Taking this into account, we used the ecological stoichiometry framework to study sexual differences in the stoichiometric phenotypes, reflecting stoichiometric niches, of four spider taxa differing in the hunting mode. The species and sexes differed fundamentally in their elemental phenotypes, including elements beyond those most commonly studied (C, N and P). Both species and sexes were distinguished by the C:N ratio and concentrations of Cu, K and Zn. Species additionally differed in concentrations of Na, Mg and Mn. Phosphorous was not involved in this differentiation. Sexual dimorphism in spiders' elemental phenotypes, related to differences in their stoichiometric niches, suggests different nutritional optima and differences in nutritional limitation experienced by different sexes and species. This may influence the structure and functioning of spider populations and communities.

The underestimated role of temperature-oxygen relationship in large-scale studies on size-to-temperature response.

The observation that ectotherm size decreases with increasing temperature (temperature-size rule; TSR) has been widely supported. This phenomenon intrigues researchers because neither its adaptive role nor the conditions under which it is realized are well defined. In light of recent theoretical and empirical studies, oxygen availability is an important candidate for understanding the adaptive role behind TSR. However, this hypothesis is still undervalued in TSR studies at the geographical level. We reanalyzed previously published data about the TSR pattern in diatoms sampled from Icelandic geothermal streams, which concluded that diatoms were an exception to the TSR. Our goal was to incorporate oxygen as a factor in the analysis and to examine whether this approach would change the results. Specifically, we expected that the strength of size response to cold temperatures would be different than the strength of response to hot temperatures, where the oxygen limitation is strongest. By conducting a regression analysis for size response at the community level, we found that diatoms from cold, well-oxygenated streams showed no size-to-temperature response, those from intermediate temperature and oxygen conditions showed reverse TSR, and diatoms from warm, poorly oxygenated streams showed significant TSR. We also distinguished the roles of oxygen and nutrition in TSR. Oxygen is a driving factor, while nutrition is an important factor that should be controlled for. Our results show that if the geographical or global patterns of TSR are to be understood, oxygen should be included in the studies. This argument is important especially for predicting the size response of ectotherms facing climate warming.

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.

Effect of combined microbes on plant tolerance to Zn-Pb contaminations.

The presence and composition of soil microbial communities has been shown to have a large impact on plant-plant interactions and consequently plant diversity and composition. The goal of the present study was to evaluate impact of arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria, which constitutes an essential link between the soil and the plant's roots. A greenhouse pot experiment was conducted to evaluate the feasibility of using selected microbes to improve Hieracium pilosella and Medicago sativa growth on Zn-Pb-rich site. Results of studies revealed that biomass, the dry mass of shoots and roots, increased significantly when plants were inoculated with mycorrhizal fungi and nitrogen-fixing bacteria. The addition of Azospirillum sp. and Nostoc edaphicum without mycorrhiza suppressed plant growth. Single bacterial inoculation alone does not have a positive effect on M. sativa growth, while co-inoculation with AMF improved plant growth. Plant vitality (expressed by the performance index) was improved by the addition of microbes. However, our results indicated that even dry heat sterilization of the substratum created imbalanced relationships between soil-plant and plants and associated microorganisms. The studies indicated that AMF and N2-fixers can improve revegetation of heavy metal-rich industrial sites, if the selection of interacting symbionts is properly conducted.

The effect of pre-analytical treatment on the results of stoichiometric measurements in invertebrates.

Growing interest in the application of stoichiometric approaches to community ecology has resulted in an increasing number of studies examining invertebrate body composition. Our experiments demonstrate various sources of possible error related to the use of pre-analytical procedures. We examined the effects of different preservatives (ethanol and formaldehyde) used in pitfall traps, time of preservation (2 weeks or 3 days) and drying method (vacuum drying at 50 °C and freeze-drying) on the determination of body composition in invertebrates representing taxa often used in such studies: earthworms and five species of insects (adults or larvae). The contents of C, N, S, P, Fe, Zn, Cu, Mn, Ca, Mg and K in each animal were measured. The use of solvents (ethanol or formaldehyde) in pitfall traps and for preservation significantly affects the body composition and stoichiometry of earthworms, even during short exposure times. Insects (both adults and larvae) were affected only during a 2-week exposure; 3 days of exposure did not significantly change their chemical composition. Vacuum-oven drying of animals at 50 °C does not affect their body composition relative to freeze-drying.