James S. Waters

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

BackgroundDespite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage.ResultsWe evaluated the tradeoff between image quality and harm to the animal by determining the impact of targeted synchrotron x-rays on insect physiology, behavior and survival. Using 25 keV x-rays at a flux density of 80 μW/mm-2, high quality video-rate images can be obtained without major detrimental effects on the insects for multiple minutes, a duration sufficient for many physiological studies. At this setting, insects do not heat up. Additionally, we demonstrate the range of uses of synchrotron phase-contrast imaging by showing high-resolution images of internal anatomy and observations of labeled food movement during ingestion and digestion.ConclusionSynchrotron x-ray phase contrast imaging has the potential to revolutionize the study of physiology and internal biomechanics in small animals. This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of living animals on the scale from millimeters to microns.

Allometric scaling of metabolism, growth, and activity in whole colonies of the seed-harvester ant Pogonomyrmex californicus

The negative allometric scaling of metabolic rate with body size is among the most striking patterns in biology. We investigated whether this pattern extends to physically independent eusocial systems by measuring the metabolic rates of whole functioning colonies of the seed‐harvester ant Pogonomyrmex californicus. These intraspecific scaling data were compared to the predictions of an additive model developed to estimate collective metabolic rates. Contrary to the prediction of the additive model, colony metabolic rate allometry resembled the pattern commonly observed interspecifically for individual organisms, scaling with colony mass0.75. Among the same‐aged colonies, net growth rate varied by up to sevenfold, with larger colonies exhibiting higher net growth efficiency than smaller colonies. Isolated worker groups exhibited isometric metabolic rate scaling, suggesting that the social environment of the colony is critical to regulating individual patterns of work output. Within the social environment, individual worker locomotor velocities exhibited power‐law distributions that scaled with colony size so that larger colonies exhibited a greater disparity between active and inactive ants than did smaller colonies. These results demonstrate that behavioral organization within colonies may have a major influence on colony‐level metabolism and in generating intraspecific variation in growth trajectories.

Basketball Teams as Strategic Networks

We asked how team dynamics can be captured in relation to function by considering games in the first round of the NBA 2010 play-offs as networks. Defining players as nodes and ball movements as links, we analyzed the network properties of degree centrality, clustering, entropy and flow centrality across teams and positions, to characterize the game from a network perspective and to determine whether we can assess differences in team offensive strategy by their network properties. The compiled network structure across teams reflected a fundamental attribute of basketball strategy. They primarily showed a centralized ball distribution pattern with the point guard in a leadership role. However, individual play-off teams showed variation in their relative involvement of other players/positions in ball distribution, reflected quantitatively by differences in clustering and degree centrality. We also characterized two potential alternate offensive strategies by associated variation in network structure: (1) whether teams consistently moved the ball towards their shooting specialists, measured as “uphill/downhill” flux, and (2) whether they distributed the ball in a way that reduced predictability, measured as team entropy. These network metrics quantified different aspects of team strategy, with no single metric wholly predictive of success. However, in the context of the 2010 play-offs, the values of clustering (connectedness across players) and network entropy (unpredictability of ball movement) had the most consistent association with team advancement. Our analyses demonstrate the utility of network approaches in quantifying team strategy and show that testable hypotheses can be evaluated using this approach. These analyses also highlight the richness of basketball networks as a dataset for exploring the relationships between network structure and dynamics with team organization and effectiveness.

How Locusts Breathe

Insect tracheal-respiratory systems achieve high fluxes and great dynamic range with low energy requirements and could be important models for bioengineers interested in developing microfluidic systems. Recent advances suggest that insect cardiorespiratory systems have functional valves that permit compartmentalization with segment-specific pressures and flows and that system anatomy allows regional flows. Convection dominates over diffusion as a transport mechanism in the major tracheae, but Reynolds numbers suggest viscous effects remain important.

Information Processing in Social Insect Networks

Investigating local-scale interactions within a network makes it possible to test hypotheses about the mechanisms of global network connectivity and to ask whether there are general rules underlying network function across systems. Here we use motif analysis to determine whether the interactions within social insect colonies resemble the patterns exhibited by other animal associations or if they exhibit characteristics of biological regulatory systems. Colonies exhibit a predominance of feed-forward interaction motifs, in contrast to the densely interconnected clique patterns that characterize human interaction and animal social networks. The regulatory motif signature supports the hypothesis that social insect colonies are shaped by selection for network patterns that integrate colony functionality at the group rather than individual level, and demonstrates the utility of this approach for analysis of selection effects on complex systems across biological levels of organization.

Critical PO2 is size-independent in insects: implications for the metabolic theory of ecology

Insects, and all animals, exhibit hypometric scaling of metabolic rate, with larger species having lower mass-specific metabolic rates. The metabolic theory of ecology (MTE) is based on models ascribing hypometric scaling of metabolic rate to constrained O2 supply systems in larger animals. We compiled critical PO2 of metabolic and growth rates for more than 40 insect species with a size range spanning four orders of magnitude. Critical PO2 values vary from far below 21kPa for resting animals to near 21kPa for growing or flying animals and are size-independent, demonstrating that supply capacity matches oxygen demand. These data suggest that hypometric scaling of resting metabolic rate in insects is not driven by constraints on oxygen availability.

Dynamics of tracheal compression in the horned passalus beetle.

Rhythmic patterns of compression and reinflation of the thin-walled hollow tubes of the insect tracheal system have been observed in a number of insects. These movements may be important for facilitating the transport and exchange of respiratory gases, but observing and characterizing the dynamics of internal physiological systems within live insects can be challenging due to their size and exoskeleton. Using synchrotron X-ray phase-contrast imaging, we observed dynamical behavior in the tracheal system of the beetle, Odontotaenius disjunctus. Similar to observations of tracheal compression in other insects, specific regions of tracheae in the thorax of O. disjunctus exhibit rhythmic collapse and reinflation. During tracheal compression, the opposing sides of a tracheal tube converge, causing the effective diameter of the tube to decrease. However, a unique characteristic of tracheal compression in this species is that certain tracheae collapse and reinflate with a wavelike motion. In the dorsal cephalic tracheae, compression begins anteriorly and continues until the tube is uniformly flattened; reinflation takes place in the reverse direction, starting with the posterior end of the tube and continuing until the tube is fully reinflated. We report the detailed kinematics of this pattern as well as additional observations that show tracheal compression coordinated with spiracle opening and closing. These findings suggest that tracheal compression may function to drive flow within the body, facilitating internal mixing of respiratory gases and ventilation of distal regions of the tracheal system.

Theoretical and empirical perspectives on the scaling of supply and demand in social insect colonies

One of the central questions in physiological ecology is how energetic constraints affect organismal performance and the dynamics of ecological systems. Social insect colonies integrate the balance of supply and demand across levels of biological organization such that the individual components are simultaneously serving as the supply transport network and also the source of energetic demand. An increasing number of studies have demonstrated that the per‐capita metabolic rates of individuals within social insect colonies decrease with increasing colony size, a metabolic hypometry much like the pattern exhibited by individual organisms. An important question is thus, whether this scaling pattern is a result of an energetic supply constraint or evidence for an emergent economy of scale. This review synthesizes theoretical models and results from empirical studies on the scaling of resource supply and demand in social insect colonies. Scaling in biology is a powerful tool to unify the study of diverse concepts and organisms; increased integration of mechanistic realism into metabolic models will improve our understanding of the evolution of complex biological systems.

Differentiating causality and correlation in allometric scaling: ant colony size drives metabolic hypometry

Metabolic rates of individual animals and social insect colonies generally scale hypometrically, with mass-specific metabolic rates decreasing with increasing size. Although this allometry has wide ranging effects on social behaviour, ecology and evolution, its causes remain controversial. Because it is difficult to experimentally manipulate body size of organisms, most studies of metabolic scaling depend on correlative data, limiting their ability to determine causation. To overcome this limitation, we experimentally reduced the size of harvester ant colonies (Pogonomyrmex californicus) and quantified the consequent increase in mass-specific metabolic rates. Our results clearly demonstrate a causal relationship between colony size and hypometric changes in metabolic rate that could not be explained by changes in physical density. These findings provide evidence against prominent models arguing that the hypometric scaling of metabolic rate is primarily driven by constraints on resource delivery or surface area/volume ratios, because colonies were provided with excess food and colony size does not affect individual oxygen or nutrient transport. We found that larger colonies had lower median walking speeds and relatively more stationary ants and including walking speed as a variable in the mass-scaling allometry greatly reduced the amount of residual variation in the model, reinforcing the role of behaviour in metabolic allometry. Following the experimental size reduction, however, the proportion of stationary ants increased, demonstrating that variation in locomotory activity cannot solely explain hypometric scaling of metabolic rates in these colonies. Based on prior studies of this species, the increase in metabolic rate in size-reduced colonies could be due to increased anabolic processes associated with brood care and colony growth.

Developmental plasticity and stability in the tracheal networks supplying Drosophila flight muscle in response to rearing oxygen level

While it is clear that the insect tracheal system can respond in a compensatory manner to both hypoxia and hyperoxia, there is substantial variation in how different parts of the system respond. However, the response of tracheal structures, from the tracheoles to the largest tracheal trunks, have not been studied within one species. In this study, we examined the effect of larval/pupal rearing in hypoxia, normoxia, and hyperoxia (10, 21 or 40kPa oxygen) on body size and the tracheal supply to the flight muscles of Drosophila melanogaster, using synchrotron radiation micro-computed tomography (SR-µCT) to assess flight muscle volumes and the major tracheal trunks, and confocal microscopy to assess the tracheoles. Hypoxic rearing decreased thorax length whereas hyperoxic-rearing decreased flight muscle volumes, suggestive of negative effects of both extremes. Tomography at the broad organismal scale revealed no evidence for enlargement of the major tracheae in response to lower rearing oxygen levels, although tracheal size scaled with muscle volume. However, using confocal imaging, we found a strong inverse relationship between tracheole density within the flight muscles and rearing oxygen level, and shorter tracheolar branch lengths in hypoxic-reared animals. Although prior studies of larger tracheae in other insects indicate that axial diffusing capacity should be constant with sequential generations of branching, this pattern was not found in the fine tracheolar networks, perhaps due to the increasing importance of radial diffusion in this regime. Overall, D. melanogaster responded to rearing oxygen level with compensatory morphological changes in the small tracheae and tracheoles, but retained stability in most of the other structural components of the tracheal supply to the flight muscles.