John M. VandenBrooks

Atmospheric oxygen level and the evolution of insect body size

Insects are small relative to vertebrates, possibly owing to limitations or costs associated with their blind-ended tracheal respiratory system. The giant insects of the late Palaeozoic occurred when atmospheric PO2 (aPO2) was hyperoxic, supporting a role for oxygen in the evolution of insect body size. The paucity of the insect fossil record and the complex interactions between atmospheric oxygen level, organisms and their communities makes it impossible to definitively accept or reject the historical oxygen-size link, and multiple alternative hypotheses exist. However, a variety of recent empirical findings support a link between oxygen and insect size, including: (i) most insects develop smaller body sizes in hypoxia, and some develop and evolve larger sizes in hyperoxia; (ii) insects developmentally and evolutionarily reduce their proportional investment in the tracheal system when living in higher aPO2, suggesting that there are significant costs associated with tracheal system structure and function; and (iii) larger insects invest more of their body in the tracheal system, potentially leading to greater effects of aPO2 on larger insects. Together, these provide a wealth of plausible mechanisms by which tracheal oxygen delivery may be centrally involved in setting the relatively small size of insects and for hyperoxia-enabled Palaeozoic gigantism.

Remote radio control of insect flight

We demonstrated the remote control of insects in free flight via an implantable radio-equipped miniature neural stimulating system. The pronotum mounted system consisted of neural stimulators, muscular stimulators, a radio transceiver-equipped microcontroller and a microbattery. Flight initiation, cessation and elevation control were accomplished through neural stimulus of the brain which elicited, suppressed or modulated wing oscillation. Turns were triggered through the direct muscular stimulus of either of the basalar muscles. We characterized the response times, success rates, and free-flight trajectories elicited by our neural control systems in remotely controlled beetles. We believe this type of technology will open the door to in-flight perturbation and recording of insect flight responses.

A cyborg beetle: Insect flight control through an implantable, tetherless microsystem

We present an implantable flight control microsystem for a cyborg beetle. The system consists of multiple inserted neural and muscular stimulators, a visual stimulator, a polyimide assembly and a microcontroller. The system is powered by two size 5 cochlear microbatteries. The insect platform is Cotinis texana, a 2 cm long, 1-2 gram Green June Beetle. We also provide data on the implantation of silicon neural probes, silicon chips, microfluidic tubes, and LEDs introduced during the pupal stage of the beetle.

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.

Oxygen supply limits the heat tolerance of lizard embryos

The mechanisms that set the thermal limits to life remain uncertain. Classically, researchers thought that heating kills by disrupting the structures of proteins or membranes, but an alternative hypothesis focuses on the demand for oxygen relative to its supply. We evaluated this alternative hypothesis by comparing the lethal temperature for lizard embryos developing at oxygen concentrations of 10–30%. Embryos exposed to normoxia and hyperoxia survived to higher temperatures than those exposed to hypoxia, suggesting that oxygen limitation sets the thermal maximum. As all animals pass through an embryonic stage where respiratory and cardiovascular systems must develop, oxygen limitation may limit the thermal niches of terrestrial animals as well as aquatic ones.

Caterpillars selected for large body size and short development time are more susceptible to oxygen-related stress

Recent studies suggest that higher growth rates may be associated with reduced capacities for stress tolerance and increased accumulated damage due to reactive oxygen species. We tested the response of Manduca sexta (Sphingidae) lines selected for large or small body size and short development time to hypoxia (10 kPa) and hyperoxia (25, 33, and 40 kPa); both hypoxia and hyperoxia reduce reproduction and oxygen levels over 33 kPa have been shown to increase oxidative damage in insects. Under normoxic (21 kPa) conditions, individuals from the large-selected (big-fast) line were larger and had faster growth rates, slightly longer developmental times, and reduced survival rates compared to individuals from a line selected for small size (small-fast) or an unselected control line. Individuals from the big-fast line exhibited greater negative responses to hyperoxia with greater reductions in juvenile and adult mass, growth rate, and survival than the other two lines. Hypoxia generally negatively affected survival and growth/size, but the lines responded similarly. These results are mostly consistent with the hypothesis that simultaneous acquisition of large body sizes and short development times leads to reduced capacities for coping with stressful conditions including oxidative damage. This result is of particular importance in that natural selection tends to decrease development time and increase body size.

Acute and chronic effects of atmospheric oxygen on the feeding behavior of Drosophila melanogaster larvae.

All insects studied to date show reduced growth rates in hypoxia. Drosophila melanogaster reared in moderate hypoxia (10 kPa PO2) grow more slowly and form smaller adults, but the mechanisms responsible are unclear, as metabolic rates are not oxygen-limited. It has been shown that individual fruit flies do not grow larger in hyperoxia (40 kPa PO2), but populations of flies evolve larger size. Here we studied the effect of acute and chronic variation in atmospheric PO2 (10, 21, 40 kPa) on feeding behavior of third instar larvae of D.melanogaster to assess whether oxygen effects on growth rate can be explained by effects on feeding behavior. Hypoxic-reared larvae grew and developed more slowly, and hyperoxic-rearing did not affect growth rate, maximal larval mass or developmental time. The effect of acute exposure to varying PO2 on larval bite rates matched the pattern observed for growth rates, with a 22% reduction in 10 kPa PO2 and no effect of 40 kPa PO2. Chronic rearing in hypoxia had few effects on the responses of feeding rates to oxygen, but chronic rearing in hyperoxia caused feeding rates to be strongly oxygen-dependent. Hypoxia produced similar reductions in bite rate and in the volume of tunnels excavated by larvae, supporting bite rate as an index of feeding behavior. Overall, our data show that reductions in feeding rate can explain reduced growth rates in moderate hypoxia for Drosophila, contributing to reduced body size, and that larvae cannot successfully compensate for this level of hypoxia with developmental plasticity.

A positive genetic correlation between hypoxia tolerance and heat tolerance supports a controversial theory of heat stress

We used quantitative genetics to test a controversial theory of heat stress, in which animals overheat when the demand for oxygen exceeds the supply. This theory, referred to as oxygen- and capacity-limited thermal tolerance, predicts a positive genetic correlation between hypoxia tolerance and heat tolerance. We demonstrate the first genetic correlation of this kind in a model organism, Drosophila melanogaster. Genotypes more likely to fly under hypoxic stress (12% O2) were also more likely to fly under heat stress (39°C). This finding prompts new questions about mechanisms and limits of adaptation to heat stress.

Developmental plasticity evolved according to specialist–generalist trade-offs in experimental populations of Drosophila melanogaster

We studied the evolution of developmental plasticity in populations of Drosophila melanogaster that evolved at either constant or fluctuating temperatures. Consistent with theory, genotypes that evolved at a constant 16°C or 25°C performed best when raised and tested at that temperature. Genotypes that evolved at fluctuating temperatures performed well at either temperature, but only when raised and tested at the same temperature. Our results confirm evolutionary patterns predicted by theory, including a loss of plasticity and a benefit of specialization in constant environments.

More oxygen during development enhanced flight performance but not thermal tolerance of Drosophila melanogaster

High temperatures can stress animals by raising the oxygen demand above the oxygen supply. Consequently, animals under hypoxia could be more sensitive to heating than those exposed to normoxia. Although support for this model has been limited to aquatic animals, oxygen supply might limit the heat tolerance of terrestrial animals during energetically demanding activities. We evaluated this model by studying the flight performance and heat tolerance of flies (Drosophila melanogaster) acclimated and tested at different concentrations of oxygen (12%, 21%, and 31%). We expected that flies raised at hypoxia would develop into adults that were more likely to fly under hypoxia than would flies raised at normoxia or hyperoxia. We also expected flies to benefit from greater oxygen supply during testing. These effects should have been most pronounced at high temperatures, which impair locomotor performance. Contrary to our expectations, we found little evidence that flies raised at hypoxia flew better when tested at hypoxia or tolerated extreme heat better than did flies raised at normoxia or hyperoxia. Instead, flies raised at higher oxygen levels performed better at all body temperatures and oxygen concentrations. Moreover, oxygen supply during testing had the greatest effect on flight performance at low temperature, rather than high temperature. Our results poorly support the hypothesis that oxygen supply limits performance at high temperatures, but do support the idea that hyperoxia during development improves performance of flies later in life.