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'.

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.

Thinking "outside the box": The effect of nontarget snails in the aquatic community on mollusc-borne diseases.

There is a great need to understand the impact of complex communities on the free-living parasite stages that are part of them. This task becomes more complex as nonnative species emerge, changing existing relationships and shaping new interactions in the community. A relevant question would be: Can the coexistence of nontarget snails with the target hosts contribute to trematodasis control? We used field and experimental approaches to investigate nonnative competitor-induced parasite dilution. During a three-year field study, we investigated digenean infection in Lymnaea stagnalis from eight Polish lakes inhabited or uninhabited by Potamopyrgus antipodarum. Additionally, we verified the presence of digenean infections in the populations of P. antipodarum. Moreover, we conducted an experimental infection of L. stagnalis with miracidia of Trichobilharzia szidati under increasing densities of P. antipodarum and aimed to infect P. antipodarum with them separately. The prevalence of avian schistosomes in lymnaeid snails was significantly higher in uninhabited lakes than in lakes inhabited by P. antipodarum. Our study indicates that waters with a higher density of invaders have a lower prevalence of avian schistosomes in lymnaeid hosts. The results of experimental studies confirmed that the presence of high densities of P. antipodarum reduces the probability of target host infection. Both field and experimental studies rule out the role of P. antipodarum as a source of avian schistosome cercariae. Here, a nonnative species was tested as a diluter, which in practice may be harmful to the local environment. This work is not a call for the introduction of nonnative species; it is intended to be a stimulus for researchers to continue searching for natural enemies of parasites because, as our results show, they exist. Finding natural enemies to the most dangerous species of human and animal parasites that will pose no threat to the local environment could be groundbreaking.

Concerted evolution of body mass, cell size and metabolic rate among carabid beetles.

Alterations in cell number and size are apparently associated with the body mass differences between species and sexes, but we rarely know which of the two mechanisms underlies the observed variance in body mass. We used phylogenetically informed comparisons of males and females of 19 Carabidae beetle species to compare body mass, resting metabolic rate, and cell size in the ommatidia and Malpighian tubules. We found that the larger species or larger sex (males or females, depending on the species) consistently possessed larger cells in the two tissues, indicating organism-wide coordination of cell size changes in different tissues and the contribution of these changes to the origin of evolutionary and sex differences in body mass. The species or sex with larger cells also exhibited lower mass-specific metabolic rates, and the interspecific mass scaling of metabolism was negatively allometric, indicating that large beetles with larger cells spent relatively less energy on maintenance than small beetles. These outcomes also support existing hypotheses about the fitness consequences of cell size changes, postulating that the low surface-to-volume ratio of large cells helps decrease the energetic demand of maintaining ionic gradients across cell membranes. Analyses with and without phylogenetic information yielded similar results, indicating that the observed patterns were not biased by shared ancestry. Overall, we suggest that natural selection does not operate on each trait independently and that the linkages between concerted cell size changes in different tissues, body mass and metabolic rate should thus be viewed as outcomes of correlational selection.

Growth patterns of the pan-European freshwater mussel, Anodonta anatina (Linnaeus, 1758) (Bivalvia: Unionidae), vary with sex and mortality in populations.

Post-maturation growth leading to indeterminate growth patterns is widespread in nature. However, its adaptive value is unclear. Life history theory suggests this allocation strategy may be favored by temporal pulses in the intensity of mortality and/or the capacity to produce new tissues.Addressing the origin of indeterminate growth and the variability of growth patterns, we studied the growth of duck mussels, Anodonta anatina, a pan-European unionid, in 18 Polish lakes. For each population, the sex, size, and age of collected mussels were measured to estimate Bertalanffy's growth curve parameters. We integrated information on A. anatina mortality rates, lake trophy, biofouling by zebra mussels, Dreissena polymorpha, and the prevalence of parasitic trematode larvae to identify selective conditions in lakes.We found two sources of mortality in A. anatina populations, pertaining to adverse effects of zebra mussel biofouling and trophy state on mussel survival. Additionally, populations with heavier biofouling presented a smaller abundance of parasites, indicative of a relationship between filtering intensity and contraction of water-borne trematode larvae by filtering A. anatina.Consistently for each sex, populations with a greater trophy-related mortality were characterized in A. anatina by a smaller asymptotic size Lmax , indicative of a life history response to mortality risk involving early maturation at a smaller body size. In all populations, females featured higher mortality and larger asymptotic size versus males.Our findings support a theoretical view that adaptive responses to selection involve adjustments in the lifetime resource allocation patterns. These adjustments should be considered drivers of the origin of indeterminate growth strategy in species taking parental care by offspring brooding in body cavities.

Effects of thermal and oxygen conditions during development on cell size in the common rough woodlice Porcellio scaber.

During development, cells may adjust their size to balance between the tissue metabolic demand and the oxygen and resource supply: Small cells may effectively absorb oxygen and nutrients, but the relatively large area of the plasma membrane requires costly maintenance. Consequently, warm and hypoxic environments should favor ectotherms with small cells to meet increased metabolic demand by oxygen supply. To test these predictions, we compared cell size (hindgut epithelium, hepatopancreas B cells, ommatidia) in common rough woodlice (Porcellio scaber) that were developed under four developmental conditions designated by two temperatures (15 or 22°C) and two air O2 concentrations (10% or 22%). To test whether small-cell woodlice cope better under increased metabolic demand, the CO2 production of each woodlouse was measured under cold, normoxic conditions and under warm, hypoxic conditions, and the magnitude of metabolic increase (MMI) was calculated. Cell sizes were highly intercorrelated, indicative of organism-wide mechanisms of cell cycle control. Cell size differences among woodlice were largely linked with body size changes (larger cells in larger woodlice) and to a lesser degree with oxygen conditions (development of smaller cells under hypoxia), but not with temperature. Developmental conditions did not affect MMI, and contrary to predictions, large woodlice with large cells showed higher MMI than small woodlice with small cells. We also observed complex patterns of sexual difference in the size of hepatopancreatic cells and the size and number of ommatidia, which are indicative of sex differences in reproductive biology. We conclude that existing theories about the adaptiveness of cell size do not satisfactorily explain the patterns in cell size and metabolic performance observed here in P. scaber. Thus, future studies addressing physiological effects of cell size variance should simultaneously consider different organismal elements that can be involved in sustaining the metabolic demands of tissue, such as the characteristics of gas-exchange organs and O2-binding proteins.

Thermal and oxygen conditions during development cause common rough woodlice (Porcellio scaber) to alter the size of their gas-exchange organs.

Terrestrial isopods have evolved pleopodal lungs that provide access to the rich aerial supply of oxygen. However, isopods occupy conditions with wide and unpredictable thermal and oxygen gradients, suggesting that they might have evolved adaptive developmental plasticity in their respiratory organs to help meet metabolic demand over a wide range of oxygen conditions. To explore this plasticity, we conducted an experiment in which we reared common rough woodlice (Porcellio scaber) from eggs to maturation at different temperatures (15 and 22 °C) combined with different oxygen levels (10% and 22% O2). We sampled animals during development (only females) and then examined mature adults (both sexes). We compared woodlice between treatments with respect to the area of their pleopod exopodites (our proxy of lung size) and the shape of Bertalanffy's equations (our proxy of individual growth curves). Generally, males exhibited larger lungs than females relative to body size. Woodlice also grew relatively fast but achieved a decreased asymptotic body mass in response to warm conditions; the oxygen did not affect growth. Under hypoxia, growing females developed larger lungs compared to under normoxia, but only in the late stage of development. Among mature animals, this effect was present only in males. Woodlice reared under warm conditions had relatively small lungs, in both developing females (the effect was increased in relatively large females) and among mature males and females. Our results demonstrated that woodlice exhibit phenotypic plasticity in their lung size. We suggest that this plasticity helps woodlice equilibrate their gas exchange capacity to differences in the oxygen supply and metabolic demand along environmental temperature and oxygen gradients. The complex pattern of plasticity might indicate the effects of a balance between water conservation and oxygen uptake, which would be especially pronounced in mature females that need to generate an aqueous environment inside their brood pouch.

Effects of Cadmium Bioavailability in Food on Its Distribution in Different Tissues in the Ground Beetle Pterostichus oblongopunctatus.

In most laboratory studies with oral exposure of terrestrial invertebrates to metals an artificial food, which is easy to handle, is used. The bioavailability of metals from this artificial food may, however, be much higher than from more field relevant food sources. Such differences may affect toxicokinetic processes in different tissues. To test the effect of bioavailability of Cd in food on Cd toxicokinetics and internal distribution in terrestrial invertebrates, we performed the experiment using the ground beetle Pterostichus oblongopunctatus exposed to Cd via food differing in their soluble Cd pool. We showed that in carabids Cd accumulation and elimination pattern in different tissues is not governed by the metal availability in food.

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.

Concerted evolution of body mass and cell size: similar patterns among species of birds (Galliformes) and mammals (Rodentia).

Cell size plays a role in body size evolution and environmental adaptations. Addressing these roles, we studied body mass and cell size in Galliformes birds and Rodentia mammals, and collected published data on their genome sizes. In birds, we measured erythrocyte nuclei and basal metabolic rates (BMRs). In birds and mammals, larger species consistently evolved larger cells for five cell types (erythrocytes, enterocytes, chondrocytes, skin epithelial cells, and kidney proximal tubule cells) and evolved smaller hepatocytes. We found no evidence that cell size differences originated through genome size changes. We conclude that the organism-wide coordination of cell size changes might be an evolutionarily conservative characteristic, and the convergent evolutionary body size and cell size changes in Galliformes and Rodentia suggest the adaptive significance of cell size. Recent theory predicts that species evolving larger cells waste less energy on tissue maintenance but have reduced capacities to deliver oxygen to mitochondria and metabolize resources. Indeed, birds with larger size of the abovementioned cell types and smaller hepatocytes have evolved lower mass-specific BMRs. We propose that the inconsistent pattern in hepatocytes derives from the efficient delivery system to hepatocytes, combined with their intense involvement in supracellular function and anabolic activity.

Effects of fat and exoskeletal mass on the mass scaling of metabolism in Carabidae beetles.

The rate at which organisms metabolize resources and consume oxygen is tightly linked to body mass. Typically, there is a sub-linear allometric relationship between metabolic rates and body mass (mass-scaling exponent b < 1). The origin of this pattern remains one of the most intriguing and hotly debated topics in evolutionary physiology. A decrease in mass-specific metabolic rates in larger organisms might reflect disproportionate increases in body components with low metabolic activity, such as storage and skeletal tissues. Addressing this hypothesis, we studied standard metabolic rates, body mass, and fat and exoskeletal mass in males and females from 15 species of Carabidae beetles. There was a sub-linear allometric relationship of metabolic rate with body mass: b = 0.72 (phylogeny not considered), b = 0.54 (phylogeny considered). The latter exponent was significantly lower than 0.75, which is sometimes regarded as a universal exponent value in the mass scaling of metabolic rates. Contrary to our hypothesis, the relative contribution of fat and the exoskeleton to body mass decreased, rather than increased with body mass, as indicated by the sub-linear allometric mass scaling of both components (b < 1). Supporting the role of metabolically inert body components in shaping metabolic scaling, the exponents (b) for metabolism became slightly smaller (b = 0.70, phylogeny not considered; 0.52, phylogeny considered) when we removed lipids and the exoskeleton from body mass calculations and considered only the lean mass of soft tissue in the mass scaling. Overall, our results indicate that, in beetles, the relative content of metabolically inert components changes across species according to species-specific body mass. Nevertheless, we did not find evidence that this changing contribution plays a central role in the origin of interspecific metabolic scaling in carabids. Our findings stress the need for finding alternative explanations, at least in carabids, for the origin of the mass scaling of metabolic rates.

Not all cells are equal: effects of temperature and sex on the size of different cell types in the Madagascar ground gecko Paroedura picta.

Cell size plays a role in evolutionary and phenotypically plastic changes in body size. To examine this role, we measured the sizes of seven cell types of geckos (Paroedura picta) reared at three constant temperatures (24, 27, and 30°C). Our results show that the cell size varies according to the body size, sex and developmental temperature, but the pattern of this variance depends on the cell type. We identified three groups of cell types, and the cell sizes changed in a coordinated manner within each group. Larger geckos had larger erythrocytes, striated muscle cells and hepatocytes (our first cell group), but their renal proximal tubule cells and duodenal enterocytes (our second cell group), as well as tracheal chondrocytes and epithelial skin cells (our third cell group), were largely unrelated to the body size. For six cell types, we also measured the nuclei and found that larger cells had larger nuclei. The relative sizes of the nuclei were not invariant but varied in a complex manner with temperature and sex. In conclusion, we provide evidence suggesting that changes in cell size might be commonly involved in the origin of thermal and sexual differences in adult size. A recent theory predicts that smaller cells speed up metabolism but demand more energy for their maintenance; consequently, the cell size matches the metabolic demand and supply, which in ectotherms, largely depends on the thermal conditions. The complex thermal dependency of cell size in geckos suggests that further advancements in understanding the adaptive value of cell size requires the consideration of tissue-specific demand/supply conditions.

Hindlimb muscle fibre size and glycogen stores in bank voles with increased aerobic exercise metabolism.

To test hypotheses concerning physiological factors limiting the rate of aerobic exercise metabolism, we used a unique experimental evolution model: lines of bank voles selected for high swim-induced aerobic metabolism (A) and unselected, control lines (C). We investigated putative adaptations that result in the increased performance of the hindlimb muscle (gastrocnemius joined with plantaris). The body mass-adjusted muscle mass was higher in A-lines (0.093 g) than in C-lines (0.083 g; P=0.01). However, selection did not affect mean muscle fibre cross-sectional area (P=0.34) or glycogen content assessed with a histochemical periodic acid-Schiff reaction (PAS; P=0.82). The results suggest that the increased aerobic performance is achieved by an increase of total muscle mass, without major qualitative changes in the muscle fibre architecture. However, such a conclusion should be treated with caution, because other modifications, such as increased density of capillaries or mitochondria, could occur.

Hold your breath beetle-Mites!

Respiratory gas exchange in insects occurs via a branching tracheal system. The entrances to the air-filled tracheae are the spiracles, which are gate-like structures in the exoskeleton. The open or closed state of spiracles defines the three possible gas exchange patterns of insects. In resting insects, spiracles may open and close over time in a repeatable fashion that results in a discontinuous gas exchange (DGE) pattern characterized by periods of zero organism-to-environment gas exchange. Several adaptive hypotheses have been proposed to explain why insects engage in DGE, but none have attracted overwhelming support. We provide support for a previously untested hypothesis that posits that DGE minimizes the risk of infestation of the tracheal system by mites and other agents. Here, we analyze the respiratory patterns of 15 species of ground beetle (Carabidae), of which more than 40% of individuals harbored external mites. Compared with mite-free individuals, infested one's engaged significantly more often in DGE. Mite-free individuals predominantly employed a cyclic or continuous gas exchange pattern, which did not include complete spiracle closure. Complete spiracle closure may prevent parasites from invading, clogging, or transferring pathogens to the tracheal system or from foraging on tissue not protected by thick chitinous layers.

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.

Automated measurement of ommatidia in the compound eyes of beetles.

The size of the ommatidia that compose the insect compound eye is linked to visual capacity, physiological performance, and cell size. Therefore, rapid and reliable methods for measuring ommatidia can advance research on insect ecology and evolution. We developed an automated method to measure ommatidia in nail polish imprints of the eyes of three Carabidae beetle species using the widely available, free software ImageJ. Our automated method was equivalent to a traditional manual method in terms of accuracy but had the advantage of being 70 times faster. We provide access to our algorithm, which can be used to investigate biological phenomena ranging from the functional architecture of the compound eye to the cellular basis of the evolution of body size and metabolic rates.

Does temperature and oxygen affect duration of intramarsupial development and juvenile growth in the terrestrial isopod Porcellioscaber (Crustacea, Malacostraca)?

According to the temperature-size rule (TSR), ectotherms developing under cold conditions experience slower growth as juveniles but reach a larger size at maturity. Whether temperature alone causes this phenomenon is unknown, but oxygen limitation can play a role in the temperature-size relationship. Oxygen may become limited under warm conditions when the resulting higher metabolism creates a greater demand for oxygen, especially in larger individuals. We examined the independent effects of oxygen concentration (10% and 22% O2) and temperature (15 °C and 22 °C) on duration of ontogenic development, which takes place within the maternal brood pouch (marsupium), and juvenile growth in the terrestrial isopod common rough woodlouse (Porcellioscaber). Individuals inside the marsupium undergo the change from the aqueous to the gaseous environment. Under hypoxia, woodlice hatched from the marsupium sooner, but their subsequent growth was not affected by the level of oxygen. Marsupial development and juvenile growth were almost three times slower at low temperature, and marsupial development was longer in larger females but only in the cold treatment. These results show that temperature and oxygen are important ecological factors affecting developmental time and that the strength of the effect likely depends on the availability of oxygen in the environment.

Effect of the abrasive properties of sedges on the intestinal absorptive surface and resting metabolic rate of root voles.

Recent studies on grasses and sedges suggest that the induction of a mechanism reducing digestibility of plant tissues in response to herbivore damage may drive rodent population cycles. This defence mechanism seems to rely on the abrasive properties of ingested plants. However, the underlying mechanism has not been demonstrated in small wild herbivores. Therefore, we carried out an experiment in which we determined the joint effect of abrasive sedge components on the histological structure of small intestine as well as resting metabolic rate (RMR) of the root vole (Microtus oeconomus). Histological examination revealed that voles fed with a sedge-dominated diet had shorter villi composed from narrower enterocytes in duodenum, jejunum and ileum. Reduction in the height of villi decreased along the small intestine. Activity of the mucus secretion increased along the small intestine and was significantly higher in the ileum. The intestinal abrasion exceeded the compensatory capabilities of voles, which responded to a sedge-dominated diet by a reduction of body mass and a concomitant decrease in whole body RMR. These results explain the inverse association between body mass and the probability of winter survival observed in voles inhabiting homogenous sedge wetlands.

Cytogenetic and morphological characterization of Corbicula fluminalis (O. F. Müller, 1774) (Bivalvia: Veneroida: Corbiculidae): taxonomic status assessment of a freshwater clam.

Chromosomes of Corbicula fluminalis were characterized by karyotype analysis and nucleolar organizer region (NORs) localization. The triploid chromosome number was confirmed as 54; the karyotype is composed of 3 metacentric, 15 submetacentric and 36 subtelo-acrocentic chromosomes. Silver staining revealed nucleolar organizers on the telomeric regions of three subtelo-acrocentic chromosomes. This is the first study on chromosomes of C. fluminalis. The results are discussed with regards to Corbicula species as well as its relationships to other mollusc species based on cytogenetic characters and morphometric of the shells.