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.

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.

Heat stress during development makes antlion larvae more responsive to vibrational cues.

We investigated the effects of heat stress on the responsiveness to vibrational cues, our measure of perceptual ability, in Myrmeleon bore antlion larvae (Neuroptera: Myrmeleontidae). We reared these trap-building predatory larvae under 2 heat stress regimes (mild, 30°C, and harsh, 36°C), and after they progressed from one instar stage to another, we tested their perceptual ability in common unchallenging conditions. We hypothesized that exposure to the harsh heat stress regime would impose costs resulting in handicapped vibration responsiveness. We found that the harsh heat stress regime generated more stressful conditions for the larvae, as evidenced by increased mortality and postponed molting, and the loss of body mass among larger larvae. Furthermore, among the individuals who remained alive, those originating from the harsh heat stress regime were characterized by higher vibration responsiveness. Our results suggest 2 not mutually exclusive scenarios. Costly heat stress conditions can sieve out individuals characterized by poor perceptual ability or surviving individuals can attempt to hunt more efficiently to compensate for the physiological imbalance caused by heat stress. Both of these mechanisms fit into the ongoing debate over how adaptation and plasticity contribute to shaping insect communities exposed to heat stress.

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.

Past thermal conditions affect hunting behaviour in larval antlions.

Some sit-and-wait predators, such as antlion larvae, construct traps to capture passing prey. The location of these traps depends on many abiotic and biotic factors, including temperature and the presence of conspecifics, which probably stimulate behaviours that minimize the costs and maximize the benefits of trap building. Here, we exposed second instar antlion larvae to elevated temperatures of 25°C (mild treatment) or 31°C (harsh treatment) for one month and then transferred them to common conditions (20°C) to examine the effects of previous thermal treatment on aggregation tendency and trap size. We predicted that antlions that experienced harsh conditions would subsequently increase the neighbouring distance and trap diameter to reduce competition with conspecifics and improve prey capture success, compensating for past conditions. In contrast with these predictions, antlions exposed to harsh conditions displayed a trend in the opposite direction, towards the decreased neighbouring distance. Furthermore, some of these antlions also built smaller traps. We discuss possible reasons for our results. The effects of previous thermal exposure have rarely been considered in terms of trap construction in antlions. Described effects may possibly apply to other sit-and-wait predators and are significant considering that many of these predators are long-lived.

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.

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.

Thermal Preferences of Cowpea Seed Beetles (Callosobruchus maculatus): Effects of Sex and Nuptial Gift Transfers.

The thermal environment influences insect performance, but the factors affecting insect thermal preferences are rarely studied. We studied Callosobruchus maculatus seed beetles and hypothesized that thermal preferences are influenced by water balance, with individuals with limited water reserves preferring cooler habitats to reduce evaporative water loss. Adult C. maculatus, in their flightless morph, do not consume food or water, but a copulating male provides a female with a nuptial gift of ejaculate containing nutrients and water. We hypothesized that gift recipients would prefer warmer habitats than gift donors and that both sexes would plastically adjust their thermal preferences according to the size of the transferred gift. We measured the thermal preference in each sex in individuals that were mated once or were unmated. In the mated group, we measured the sizes of the nuptial gifts and calculated proportional body mass changes in each mate during copulation. Supporting the role of water balance in thermal preference, females preferred warmer habitats than males. Nevertheless, thermal preferences in either sex were not affected by mating status or gift size. It is likely that high rates of mating and gift transfers in C. maculatus living under natural conditions promoted the evolution of constitutive sex-dependent thermal preferences.

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.

Stoichiometric niche, nutrient partitioning and resource allocation in a solitary bee are sex-specific and phosphorous is allocated mainly to the cocoon.

Life histories of species may be shaped by nutritional limitations posed on populations. Yet, populations contain individuals that differ according to sex and life stage, each of which having different nutritional demands and experiencing specific limitations. We studied patterns of resource assimilation, allocation and excretion during the growth of the solitary bee Osmia bicornis (two sexes) under natural conditions. Adopting an ecological perspective, we assert that organisms ingest mutable organic molecules that are transformed during physiological processes and that the immutable atoms of the chemical elements composing these molecules may be allocated to specific functions, thereby influencing organismal fitness and life history. Therefore, using the framework of ecological stoichiometry, we investigated the multielemental (C, N, S, P, K, Na, Ca, Mg, Fe, Zn, Mn, Cu) compositions of six components of the bee elemental budget: food (pollen), eggs, pupae, adults, cocoons and excreta. The sexes differed fundamentally in the assimilation and allocation of acquired atoms, elemental phenotypes, and stoichiometric niches for all six components. Phosphorus, which supports larval growth, was allocated mainly (55-75%) to the cocoon after larval development was complete. Additionally, the majority (60-99%) of the Mn, Ca, Mg and Zn acquired during larval development was allocated to the cocoon, probably influencing bee fitness by conferring protection. We conclude that for holometabolous insects, considering only the chemical composition of the adult body within the context of nutritional ecology does not provide a complete picture. Low ratios of C to other nutrients, low N:P and high Na concentrations in excreta and cocoons may be important for local-scale nutrient cycling. Limited access to specific nutritional elements may hinder bee development in a sex-dependent manner, and N and P limitations, commonly considered elsewhere, may not play important roles in O. bicornis. Sexual dimorphism in nutritional limitations due to nutrient scarcity during the larval stage may influence bee population function and should be considered in bee conservation efforts.

Heat wave effects on the behavior and life-history traits of sedentary antlions.

Research on the behavioral responses of animals to extreme weather events, such as heat wave, is lacking even though their frequency and intensity in nature are increasing. Here, we investigated the behavioral response to a simulated heat wave in two species of antlions (Neuroptera: Myrmeleontidae). These insects spend the majority of their lives as larvae and live in sandy areas suitable for a trap-building hunting strategy. We used larvae of Myrmeleon bore and Euroleon nostras, which are characterized by different microhabitat preferences-sunlit in the case of M. bore and shaded in the case of E. nostras. Larvae were exposed to fluctuating temperatures (40 °C for 10 h daily and 25 °C for the remaining time) or a constant temperature (25 °C) for an entire week. We found increased mortality of larvae under heat. We detected a reduction in the hunting activity of larvae under heat, which corresponded to changes in the body mass of individuals. Furthermore, we found long-term consequences of the simulated heat wave, as it prolonged the time larvae needed to molt. These effects were pronounced in the case of E. nostras but did not occur or were less pronounced in the case of M. bore, suggesting that microhabitat-specific selective pressures dictate how well antlions handle heat waves. We, thus, present results demonstrating the connection between behavior and the subsequent changes to fitness-relevant traits in the context of a simulated heat wave. These results illustrate how even closely related species may react differently to the same event.

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.

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.

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.

Human-Wildlife Conflicts in Krakow City, Southern Poland.

Efforts to reduce human-wildlife-conflict are integral to wildlife management and conservation in urban habitats. In our study, we identified the HWC situations in urban areas of Krakow city, based on animal-vehicle collisions, intrusion to property, and damages. Hot spot analysis and Moran's Index were used to identify the location of maximum potential conflict. We analysed 2512 incidents in which animals (of which 85% included mammals and 15% birds) were involved in conflict situations between 2007 and 2013. A significant seasonal variation was observed among the animals. We also identified roe deer (50.23%), red fox (22.80%) and wild boar (11.40%), as the three prominent conflicted animals. Getis-Ord Gi* analysis was used to identify spatial clusters of conflict. A significant spatial association was found in the location of clusters of hot spots in specific land-use based on Moran's Index. Hot spots of roe deer and wild boar were high in grasslands and in forest and for red fox in built-up area. The results underscore the notion that conservation and wildlife management efforts must take into account differences in the seasonality of HWC among species. This information can be used to inform mitigation strategies.

Coevolution of body size and metabolic rate in vertebrates: a life-history perspective.

Despite many decades of research, the allometric scaling of metabolic rates (MRs) remains poorly understood. Here, we argue that scaling exponents of these allometries do not themselves mirror one universal law of nature but instead statistically approximate the non-linearity of the relationship between MR and body mass. This 'statistical' view must be replaced with the life-history perspective that 'allows' organisms to evolve myriad different life strategies with distinct physiological features. We posit that the hypoallometric allometry of MRs (mass scaling with an exponent smaller than 1) is an indirect outcome of the selective pressure of ecological mortality on allocation 'decisions' that divide resources among growth, reproduction, and the basic metabolic costs of repair and maintenance reflected in the standard or basal metabolic rate (SMR or BMR), which are customarily subjected to allometric analyses. Those 'decisions' form a wealth of life-history variation that can be defined based on the axis dictated by ecological mortality and the axis governed by the efficiency of energy use. We link this variation as well as hypoallometric scaling to the mechanistic determinants of MR, such as metabolically inert component proportions, internal organ relative size and activity, cell size and cell membrane composition, and muscle contributions to dramatic metabolic shifts between the resting and active states. The multitude of mechanisms determining MR leads us to conclude that the quest for a single-cause explanation of the mass scaling of MRs is futile. We argue that an explanation based on the theory of life-history evolution is the best way forward.