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.

Rapamycin supplementation of Drosophila melanogaster larvae results in less viable adults with smaller cells.

The intrinsic sources of mortality relate to the ability to meet the metabolic demands of tissue maintenance and repair, ultimately shaping ageing patterns. Anti-ageing mechanisms compete for resources with other functions, including those involved in maintaining functional plasma membranes. Consequently, organisms with smaller cells and more plasma membranes should devote more resources to membrane maintenance, leading to accelerated intrinsic mortality and ageing. To investigate this unexplored trade-off, we reared Drosophila melanogaster larvae on food with or without rapamycin (a TOR pathway inhibitor) to produce small- and large-celled adult flies, respectively, and measured their mortality rates. Males showed higher mortality than females. As expected, small-celled flies (rapamycin) showed higher mortality than their large-celled counterparts (control), but only in early adulthood. Contrary to predictions, the median lifespan was similar between the groups. Rapamycin administered to adults prolongs life; thus, the known direct physiological effects of rapamycin cannot explain our results. Instead, we invoke indirect effects of rapamycin, manifested as reduced cell size, as a driver of increased early mortality. We conclude that cell size differences between organisms and the associated burdens of plasma membrane maintenance costs may be important but overlooked factors influencing mortality patterns in nature.

Systemic changes in cell size throughout the body of Drosophila melanogaster associated with mutations in molecular cell cycle regulators.

Along with different life strategies, organisms have evolved dramatic cellular composition differences. Understanding the molecular basis and fitness effects of these differences is key to elucidating the fundamental characteristics of life. TOR/insulin pathways are key regulators of cell size, but whether their activity determines cell size in a systemic or tissue-specific manner awaits exploration. To that end, we measured cells in four tissues in genetically modified Drosophila melanogaster (rictorΔ2 and Mnt1) and corresponding controls. While rictorΔ2 flies lacked the Rictor protein in TOR complex 2, downregulating the functions of this element in TOR/insulin pathways, Mnt1 flies lacked the transcriptional regulator protein Mnt, weakening the suppression of downstream signalling from TOR/insulin pathways. rictorΔ2 flies had smaller epidermal (leg and wing) and ommatidial cells and Mnt1 flies had larger cells in these tissues than the controls. Females had consistently larger cells than males in the three tissue types. In contrast, dorsal longitudinal flight muscle cells (measured only in males) were not altered by mutations. We suggest that mutations in cell cycle control pathways drive the evolution of systemic changes in cell size throughout the body, but additional mechanisms shape the cellular composition of some tissues independent of these mutations.

Systemic orchestration of cell size throughout the body: influence of sex and rapamycin exposure in Drosophila melanogaster.

Along with differences in life histories, metazoans have also evolved vast differences in cellularity, involving changes in the molecular pathways controlling the cell cycle. The extent to which the signalling network systemically determines cellular composition throughout the body and whether tissue cellularity is organized locally to match tissue-specific functions are unclear. We cultured genetic lines of Drosophila melanogaster on food with and without rapamycin to manipulate the activity of target of rapamycin (TOR)/insulin pathways and evaluate cell-size changes in five types of adult cells: wing and leg epidermal cells, ommatidial cells, indirect flight muscle cells and Malpighian tubule epithelial cells. Rapamycin blocks TOR multiprotein complex 1, reducing cell growth, but this effect has been studied in single cell types. As adults, rapamycin-treated flies had smaller bodies and consistently smaller cells in all tissues. Regardless, females eclosed with larger bodies and larger cells in all tissues than males. Thus, differences in TOR activity and sex were associated with the orchestration of cell size throughout the body, leading to differences in body size. We postulate that the activity of TOR/insulin pathways and their effects on cellularity should be considered when investigating the origin of ecological and evolutionary patterns in life histories.

Thermal and Oxygen Flight Sensitivity in Ageing Drosophila melanogaster Flies: Links to Rapamycin-Induced Cell Size Changes.

Ectotherms can become physiologically challenged when performing oxygen-demanding activities (e.g., flight) across differing environmental conditions, specifically temperature and oxygen levels. Achieving a balance between oxygen supply and demand can also depend on the cellular composition of organs, which either evolves or changes plastically in nature; however, this hypothesis has rarely been examined, especially in tracheated flying insects. The relatively large cell membrane area of small cells should increase the rates of oxygen and nutrient fluxes in cells; however, it does also increase the costs of cell membrane maintenance. To address the effects of cell size on flying insects, we measured the wing-beat frequency in two cell-size phenotypes of Drosophila melanogaster when flies were exposed to two temperatures (warm/hot) combined with two oxygen conditions (normoxia/hypoxia). The cell-size phenotypes were induced by rearing 15 isolines on either standard food (large cells) or rapamycin-enriched food (small cells). Rapamycin supplementation (downregulation of TOR activity) produced smaller flies with smaller wing epidermal cells. Flies generally flapped their wings at a slower rate in cooler (warm treatment) and less-oxygenated (hypoxia) conditions, but the small-cell-phenotype flies were less prone to oxygen limitation than the large-cell-phenotype flies and did not respond to the different oxygen conditions under the warm treatment. We suggest that ectotherms with small-cell life strategies can maintain physiologically demanding activities (e.g., flight) when challenged by oxygen-poor conditions, but this advantage may depend on the correspondence among body temperatures, acclimation temperatures and physiological thermal limits.

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.

Dermacentor reticulatus (Fabricius, 1794) and Babesia canis (Piana et Galli-Valerio, 1895) as the parasites of companion animals (dogs and cats) in the Wroclaw area, south-western Poland.

UNLABELLED: Tests performed in 2013 and 2014 revealed the occurrence of three tick species parasitizing pet cats and dogs in the Wroclaw Agglomeration. In total, 1,455 tick specimens were removed from 931 hosts (760 dogs and 171 cats) in 18 veterinary clinics. The dominant tick species was Ixodes ricinus (n=1272; 87.4%), followed by I. hexagonus (n=137; 9.4%) and Dermacentor reticulatus (n=46; 3.2%). Females were the most often collected development stage among I. ricinus and D. reticulatus, and nymphs among I. hexagonus. Additionally, D. reticulatus ticks (n=337) were then collected from vegetation in the Wroclaw area to detect Babesia canis; however, none was found positive. Only 9.0% of dog blood samples sent to VETLAB were positive for Babesia spp. Negative results for B. canis from ticks may result from the short period of the occurrence of D. reticulatus in the Wroclaw area and therefore the vectorpathogen cycle may not have been fully established at the time of the study. Nevertheless, D. reticulatus is expanding its range, and the size of its population in the Wroclaw Agglomeration is increasing. The presence of the pathogenic Babesia spp. combined with the occurrence of its main vector¸ D. reticulatus, suggests that the epizootiological situation in the area can change and may pose a new veterinary problem in the future. KEY WORDS: Dermacentor reticulatus, Babesia canis, pets, Wroclaw, Poland.