Taxogenomic analyses of Starmerella gilliamiae f.a, sp. nov. and Starmerella monicapupoae f.a., sp. nov., two novel species isolated from plant substrates and insects.

Four yeast isolates collected from flowers from different ecosystems in Brazil, one from fruit of Nothofagus alpina in Argentina, three from flowers of Neltuma chilensis in Chile and one obtained from the proventriculus of a female bumblebee in Canada were demonstred, by analysis of the sequences of the internal transcribed spacer (ITS) region and D1/D2 domains of the large subunit rRNA gene, to represent two novel species of the genus Starmerella. These species are described here as Starmerella gilliamiae f.a, sp. nov. (CBS 16166T; Mycobank MB 851206) and Starmerella monicapupoae f.a., sp. nov. (PYCC 8997T; Mycobank MB 851207). The results of a phylogenomic analysis using 1037 single-copy orthogroups indicated that S. gilliamiae is a member of a subclade that contains Starmerella opuntiae, Starmerella aceti and Starmerella apicola. The results also indicated that S. monicapupoae is phylogenetically related to Starmerella riodocensis. The two isolates of S. monicapupoae were obtained from flowers in Brazil and were probably vectored by insects that visit these substrates. Starmerella gilliamiae has a wide geographical distribution having been isolated in flowers from Brazil and Chile, fruit from Argentina and a bumblebee from Canada.

Isometric spiracular scaling in scarab beetles-implications for diffusive and advective oxygen transport.

The scaling of respiratory structures has been hypothesized to be a major driving factor in the evolution of many aspects of animal physiology. Here, we provide the first assessment of the scaling of the spiracles in insects using 10 scarab beetle species differing 180× in mass, including some of the most massive extant insect species. Using X-ray microtomography, we measured the cross-sectional area and depth of all eight spiracles, enabling the calculation of their diffusive and advective capacities. Each of these metrics scaled with geometric isometry. Because diffusive capacities scale with lower slopes than metabolic rates, the largest beetles measured require 10-fold higher PO2 gradients across the spiracles to sustain metabolism by diffusion compared to the smallest species. Large beetles can exchange sufficient oxygen for resting metabolism by diffusion across the spiracles, but not during flight. In contrast, spiracular advective capacities scale similarly or more steeply than metabolic rates, so spiracular advective capacities should match or exceed respiratory demands in the largest beetles. These data illustrate a general principle of gas exchange: scaling of respiratory transport structures with geometric isometry diminishes the potential for diffusive gas exchange but enhances advective capacities; combining such structural scaling with muscle-driven ventilation allows larger animals to achieve high metabolic rates when active.

Size-dependent Scaling of Stingless Bee Flight Metabolism Reveals an Energetic Benefit to Small Body Size.

Understanding the effect of body size on flight costs is critical for development of models of aerodynamics and animal energetics. Prior scaling studies that have shown that flight costs scale hypometrically have focused primarily on larger (> 100 mg) insects and birds, but most flying species are smaller. We studied the flight physiology of thirteen stingless bee species over a large range of body sizes (1-115 mg). Metabolic rate during hovering scaled hypermetrically (scaling slope = 2.11). Larger bees had warm thoraxes while small bees were nearly ecothermic; however, even controlling for body temperature variation, flight metabolic rate scaled hypermetrically across this clade. Despite having a lower mass-specific metabolic rate during flight, smaller bees could carry the same proportional load. Wingbeat frequency did not vary with body size, in contrast to most studies that find wingbeat frequency increases as body size decreases. Smaller stingless bees have greater relative forewing surface area which may help them reduce the energy requirements needed to fly. Further, we hypothesize that the relatively larger heads of smaller species may change their body pitch in flight. Synthesizing across all flying insects, we demonstrate that the scaling of flight metabolic rate changes from hypermetric to hypometric at approximately 58 mg body mass with hypermetic scaling below (slope = 1.2) and hypometric scaling (slope = 0.67) above 58 mg in body mass. The reduced cost of flight likely provides selective advantages for the evolution of small body size in insects. The biphasic scaling of flight metabolic rates and wingbeat frequencies in insects supports the hypothesis that the scaling of metabolic rate is closely related to the power requirements of locomotion and cycle frequencies.

Plasticity drives extreme cold tolerance of emerald ash borer (Agrilus planipennis) during a polar vortex.

Invasive species must often survive combinations of environmental conditions that differ considerably from their native range; however, for a given species it is unclear whether improved tolerance is the result of phenotypic plasticity or genetic adaptation (or both). Agrilus planipennis (Coleoptera: Buprestidae; the emerald ash borer) is an invasive pest of Fraxinus trees in North America and Europe. Previous studies in SW Ontario, Canada, showed that A. planipennis is freeze avoidant, preventing internal ice formation by accumulating Molar concentrations of glycerol in its hemolymph and depressing its supercooling point (SCP, the temperature at which it freezes). The cold tolerance of these SW Ontario animals was used to predict potential distribution, revealing that some Canadian cities should be too cold to allow populations to persist. However, a small population of A. planipennis has persisted in Winnipeg, Manitoba, Canada, through several severe 'polar vortex' events. In 2018/19, we collected A. planipennis larvae and prepupae from Winnipeg, MB and Southern Ontario, and found that individuals from Winnipeg were extremely cold tolerant - with SCPs as low as -52°C in prepupae (compared to -32°C in SW Ontario), and observed survival of unfrozen individuals exposed to -50°C for one hour. This cold tolerance was accompanied by higher hemolymph osmolality and glycerol concentration than in the SW Ontario individuals. To distinguish between phenotypic plasticity and local adaptation, in 2020/21 we overwintered Winnipeg-sourced individuals either outdoors in SW Ontario or in a simulated Winnipeg winter. Simulated Winnipeg winter individuals had cold tolerance similar to those overwintered in Winnipeg, while SW Ontario overwintered individuals had cold tolerance similar to those collected previously in the region. The simulated winter individuals had higher hemolymph glycerol concentrations than SW Ontario overwintered animals, at least in part due to greater dehydration. Thus, A. planipennis are cold-tolerant enough to survive some of the harshest winters where their host trees can grow, and most likely attain this cold tolerance via phenotypic plasticity. These findings raise the importance of delineating sensitivity of conclusions to unexpected phenotypic plasticity when predicting potential distributions of new invasives or responses to climate change.

White Paper: An Integrated Perspective on the Causes of Hypometric Metabolic Scaling in Animals.

Larger animals studied during ontogeny, across populations, or across species, usually have lower mass-specific metabolic rates than smaller animals (hypometric scaling). This pattern is usually observed regardless of physiological state (e.g. basal, resting, field, maximally-active). The scaling of metabolism is usually highly correlated with the scaling of many life history traits, behaviors, physiological variables, and cellular/molecular properties, making determination of the causation of this pattern challenging. For across-species comparisons of resting and locomoting animals (but less so for across populations or during ontogeny), the mechanisms at the physiological and cellular level are becoming clear. Lower mass-specific metabolic rates of larger species at rest are due to a) lower contents of expensive tissues (brains, liver, kidneys), and b) slower ion leak across membranes at least partially due to membrane composition, with lower ion pump ATPase activities. Lower mass-specific costs of larger species during locomotion are due to lower costs for lower-frequency muscle activity, with slower myosin and Ca++ ATPase activities, and likely more elastic energy storage. The evolutionary explanation(s) for hypometric scaling remain(s) highly controversial. One subset of evolutionary hypotheses relies on constraints on larger animals due to changes in geometry with size; for example, lower surface-to-volume ratios of exchange surfaces may constrain nutrient or heat exchange, or lower cross-sectional areas of muscles and tendons relative to body mass ratios would make larger animals more fragile without compensation. Another subset of hypotheses suggests that hypometric scaling arises from biotic interactions and correlated selection, with larger animals experiencing less selection for mass-specific growth or neurolocomotor performance. A additional third type of explanation comes from population genetics. Larger animals with their lower effective population sizes and subsequent less effective selection relative to drift may have more deleterious mutations, reducing maximal performance and metabolic rates. Resolving the evolutionary explanation for the hypometric scaling of metabolism and associated variables is a major challenge for organismal and evolutionary biology. To aid progress, we identify some variation in terminology use that has impeded cross-field conversations on scaling. We also suggest that promising directions for the field to move forward include: 1) studies examining the linkages between ontogenetic, population-level, and cross-species allometries, 2) studies linking scaling to ecological or phylogenetic context, 3) studies that consider multiple, possibly interacting hypotheses, and 4) obtaining better field data for metabolic rates and the life history correlates of metabolic rate such as lifespan, growth rate and reproduction.

Physiological responses to gravity in an insect.

Gravity is one of the most ubiquitous environmental effects on living systems: Cellular and organismal responses to gravity are of central importance to understanding the physiological function of organisms, especially eukaryotes. Gravity has been demonstrated to have strong effects on the closed cardiovascular systems of terrestrial vertebrates, with rapidly responding neural reflexes ensuring proper blood flow despite changes in posture. Invertebrates possess open circulatory systems, which could provide fewer mechanisms to restrict gravity effects on blood flow, suggesting that these species also experience effects of gravity on blood pressure and distribution. However, whether gravity affects the open circulatory systems of invertebrates is unknown, partly due to technical measurement issues associated with small body size. Here we used X-ray imaging, radio-tracing of hemolymph, and micropressure measurements in the American grasshopper, Schistocerca americana, to assess responses to body orientation. Our results show that during changes in body orientation, gravity causes large changes in blood and air distribution, and that body position affects ventilation rate. Remarkably, we also found that insects show similar heart rate responses to body position as vertebrates, and contrasting with the classic understanding of open circulatory systems, have flexible valving systems between thorax and abdomen that can separate pressures. Gravitational effects on invertebrate cardiovascular and respiratory systems are likely to be widely distributed among invertebrates and to have broad influence on morphological and physiological evolution.

Muscle mass drives cost in sexually selected arthropod weapons.

Sexually selected weapons often function as honest signals of fighting ability. If poor-quality individuals produce high-quality weapons, then receivers should focus on other, more reliable signals. Cost is one way to maintain signal integrity. The costs of weapons tend to increase with relative weapon size, and thereby restrict large weapons to high-quality individuals who can produce and maintain them. Weapon cost, however, appears to be unpredictably variable both within and across taxa, and the mechanisms underlying this variation remain unclear. We suggest variation in weapon cost may result from variation in weapon composition-specifically, differences in the amount of muscle mass directly associated with the weapon. We test this idea by measuring the metabolic cost of sexually selected weapons in seven arthropod species and relating these measures to weapon muscle mass. We show that individuals with relatively large weapon muscles have disproportionately high resting metabolic rates and provide evidence that this trend is driven by weapon muscle mass. Overall, our results suggest that variation in weapon cost can be partially explained by variation in weapon morphology and that the integrity of weapon signals may be maintained by increased metabolic cost in species with relatively high weapon muscle mass.

The hidden cost of sexually selected traits: the metabolic expense of maintaining a sexually selected weapon.

Sexually selected weapons are among the most exaggerated traits in nature. Sexual selection theory frequently assumes a high cost of this exaggeration; yet, those costs are rarely measured. We know very little about the energetic resources required to maintain these traits at rest and the difference in energetic costs for the largest individuals relative to the smallest individuals. Knowledge in this area is crucial; resting metabolic rate can account for 30-40% of daily energy expenditure in wild animals. Here, we capitalized on the phenomenon of autotomy to take a unique look at weapon maintenance costs. Using Leptoscelis tricolor (Hemiptera: Coreidae), we measured CO2 production rates before and after a weapon was shed. Males in this insect species use enlarged hind femora as weapons in male-male combat, and yet can shed them readily, without regeneration, upon entrapment. We found that metabolic rate decreased by an average of 23.5% in males after leg loss and by 7.9% in females. Notably, larger males had less of a drop in metabolic rate per gram of weapon lost. Our findings suggest that sexually selected weapons contribute to a large portion of resting metabolic rate in males, but these costs do not scale in direct proportion to size; larger males can have larger weapons for a reduced metabolic cost. These energetic maintenance costs may be integral to the evolution of the allometries of sexually selected weapons, and yet they remain largely unexplored.

Feature-positive and feature-negative learning in honey bees.

Honey bees (Apis mellifera anatolica) were subjected to sequential trials where they were given the choice between a feature-positive and a feature-negative feeding plate. The 'feature' being manipulated is the presence of a single blue circle among three circles marking the location of a small sucrose reward. That is, a 'feature-negative' target had three white circles, while a 'feature-positive' target had two white circles and one blue one. Two experiments were performed. In both experiments, each bee was tested under two different reward scenarios (treatments). In the first experiment, during the feature-positive treatment bees received 4 µl of 2 mol l(-1) sucrose when choosing the feature-positive plate, but received 4 µl of saturated NaCl solution (saltwater) when choosing the feature-negative plate. During the feature-negative treatment, bees were rewarded when visiting the feature-negative plate, while visitation to the feature-positive plate only offered bees the saltwater. The second experiment was a repeat of the first except that pure water was offered instead of saltwater in the non-rewarding feeding plate. As an experimental control, a set of bees was offered sequential trials where both the feature-positive and feature-negative plates offered the sucrose reward. Bee feeding plate choice differed between the feature-positive and feature-negative treatments in both experiments. Bees favored the feeding plate type with the sucrose reward in each treatment, and never consumed the saltwater or pure water when encountered in either treatment. Further, behavior of bees during both the feature-positive and feature-negative treatments differed from that of control bees. However, neither feature-positive nor feature-negative learning reached high levels of success. Further, a feature-positive effect was seen when pure water was offered; bees learned to solve the feature-positive problem more rapidly. When we tested bees using simply the choice of blue versus white targets, where one color held the sucrose reward and the other the saltwater, a bee's fidelity to the color offering the sucrose reward quickly reached very high levels.