Nicotine Has a Therapeutic Window of Effectiveness in a Drosophila melanogaster Model of Parkinson's Disease.

Strong epidemiological evidence and studies in models of Parkinson's disease (PD) suggest that nicotine may be therapeutically beneficial in PD patients. However, a number of clinical trials utilizing nicotine in PD patients have had mixed results, indicating that either nicotine is not beneficial in PD patients, or an important aspect of nicotine therapy was absent. We hypothesized that nicotine must be administered early in the adult fly life in order to have beneficial effects. We show that continuous early nicotine administration improves both climbing and flight deficiencies present in homozygous park 25 mutant PD model Drosophila melanogaster. Using a new climbing assay, we identify several climbing deficiencies in this PD model that are improved or rescued by continuous nicotine treatment. Amongst these benefits, it appears that nicotine improves the ability of the park 25 flies to descend the climbing vial by being able to climb down more. In support of our hypothesis, we show that in order for nicotine benefits on climbing and flight to happen, nicotine administration must occur in a discrete time frame following adult fly eclosure: within one day for climbing or five days for flight. This therapeutic window of nicotine administration in this PD model fly may help to explain the lack of efficacy of nicotine in human clinical trials.

Oxygen environment and metabolic oxygen demand predictably interact to affect thermal behavior in a lizard, Sceloporus occidentalis.

The climate crisis necessitates predicting how organisms respond to changing environments, but this requires understanding the mechanisms underlying thermal tolerance. The Hierarchical Mechanisms of Thermal Limitation (HMTL) hypothesis proposes that respiratory capacity and marginal stability of proteins and membranes interact hierarchically to determine thermal performance and limits. An untested prediction of the HMTL hypothesis is that behavioral anapyrexia (i.e., reduced body temperature in hypoxia) is exacerbated when metabolic demand is high. We tested this prediction by manipulating western fence lizards' (Sceloporus occidentalis) metabolic demand and oxygen environment, then measuring selected body temperatures. Lizards with elevated metabolic demand selected lower body temperatures at higher oxygen concentrations than resting lizards, but this occurred only at oxygen concentrations <12% O2 , suggesting thermal limits are unaffected by naturally-occurring oxygen variation. Given our results and the ubiquity of behavioral anapyrexia, the HMTL hypothesis may generally explain how oxygen and temperature interactively affect reptile performance.

Anatomical self-efficacy of undergraduate students improves during a fully online biology course with at-home dissections.

Student enrollments in online college courses have grown steadily over the past decade, and college administrators expect this trend to continue or accelerate. Despite the growing popularity of online education, one major critique in the sciences is that students are not trained in the hands-on skills they may need for the workforce, graduate school, or professional school. For example, the Association of American Medical Colleges has recommended that medical schools evaluate applicants on their motor skills and observation skills, yet many online biology programs do not offer opportunities for students to develop these skills. In on-campus biology programs, students commonly develop these skills through hands-on animal dissections, but educators have struggled with how to teach dissections in an online environment. We designed a fully online undergraduate biology course that includes at-home, hands-on dissections of eight vertebrate specimens. Over three course offerings, we evaluated changes in four student outcomes: anatomical self-efficacy, confidence in laboratory skills, perceptions of support, and concerns about dissections. Here, we describe how we implemented at-home dissections in the online course and show that students taking the course gained anatomical self-efficacy and confidence in multiple laboratory skills. Based on open-ended responses, the students perceived that their experiences with the at-home dissections facilitated these gains. These results demonstrate that at-home, hands-on laboratories are a viable approach for teaching practical skills to students in fully online courses. We encourage science instructors to introduce at-home laboratories into their online courses, and we provide recommendations for instructors interested in implementing at-home laboratories.

Oxygen supply limits the chronic heat tolerance of locusts during the first instar only.

Although scientists know that overheating kills many organisms, they do not agree on the mechanism. According to one theory, referred to as oxygen- and capacity-limitation of thermal tolerance, overheating occurs when a warming organism's demand for oxygen exceeds its supply, reducing the organism's supply of ATP. This model predicts that an organism's heat tolerance should decrease under hypoxia, yet most terrestrial organisms tolerate the same amount of warming across a wide range of oxygen concentrations. This point is especially true for adult insects, who deliver oxygen through highly efficient respiratory systems. However, oxygen limitation at high temperatures may be more common during immature life stages, which have less developed respiratory systems. To test this hypothesis, we measured the effects of heat and hypoxia on the survival of South American locusts (Schistocerca cancellata) throughout development and during specific instars. We demonstrate that the heat tolerance of locusts depends on oxygen supply during the first instar but not during later instars. This finding provides further support for the idea that oxygen limitation of thermal tolerance depends on respiratory performance, especially during immature life stages.

Responses to Alteration of Atmospheric Oxygen and Social Environment Suggest Trade-Offs among Growth Rate, Life Span, and Stress Susceptibility in Giant Mealworms (Zophobas morio).

Growth rate, development time, and response to environmental stressors vary tremendously across organisms, suggesting trade-offs that are affected by evolutionary or ecological factors, but such trade-offs are poorly understood. Prior studies using artificially selected lines of Manduca sexta suggest that insects with high growth rates, long development time, and large body size are more sensitive to hypoxic or hyperoxic stresses, such as reactive oxygen species (ROS) production, but the mechanisms and specific life-history associations remain unclear. Here, we manipulated the social environment to differentiate the effects of size, growth rate, and development time on oxygen sensitivity of the giant mealworm, Zophobas morio. Crowding reduced growth rates but yielded larger adults as a result of supernumerary molts and longer development times. The juvenile performance (growth rate, development time, adult mass) of crowd-reared mealworms was less sensitive to variation in atmospheric oxygen than it was for individually reared animals, consistent with the hypothesis that high growth rates are associated with increased sensitivity to ROS. Life span in normoxia was extended by crowd rearing, perhaps due to the larger size and/or increased resources of the larger adults. Life spans of crowd-reared animals were more negatively affected by hypoxia or hyperoxia than life spans of individually reared animals, possibly due to the longer total stress exposure of crowd-reared animals. These data suggest that animals with high growth rates experience a negative trade-off of performance with greater sensitivity to stress during the juvenile phase, while animals with long development times or life spans experience a negative trade-off of greater susceptibility of life span to environmental stress.

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.

Oxygen supply limits the heat tolerance of avian embryos.

Physiologists have primarily focused on two potential explanations for heat stress in animals-the classic model of molecular stability and an alternative model of oxygen limitation. Although the classic model has widespread support, the oxygen-supply model applies to many aquatic animals and some terrestrial ones. In particular, the embryonic stage of terrestrial animals seems most susceptible to oxygen limitation because embryos acquire oxygen from the atmosphere by diffusion rather than ventilation. We report experiments confirming the two conditions of the oxygen-supply model in Japanese quail embryos, Coturnix coturnix. Hypoxia (12% O2) greatly reduced the chance of survival at 47.5°C, and hyperoxia greatly improved the chance of survival at 48.5°C. This finding expands the scope of the oxygen-supply model to a terrestrial, endothermic species, suggesting that oxygen supply generally limits the heat tolerance of embryos.

Oxygen Limitation Does Not Drive the Decreasing Heat Tolerance of Grasshoppers during Development.

Thermal physiology changes as organisms grow and develop, but we do not understand what causes these ontogenetic shifts. According to the theory of oxygen- and capacity-limited thermal tolerance, an organism's heat tolerance should change throughout ontogeny as its ability to deliver oxygen varies. As insects grow during an instar, their metabolic demand increases without a proportional increase in the size of tracheae that supply oxygen to the tissues. If oxygen delivery limits heat tolerance, the mismatch between supply and demand should make insects more susceptible to heat and hypoxia as they progress through an instar. We tested this hypothesis by measuring the heat tolerance of grasshoppers (Schistocerca americana) on the second and seventh days of the sixth instar, in either a normoxic or a hypoxic atmosphere (21% or 10% O2, respectively). As expected, heat tolerance decreased as grasshoppers grew larger. Yet contrary to expectation, hypoxia had no effect on heat tolerance across all stages and sizes. Although heat tolerance declines as grasshoppers grow, this pattern must stem from a mechanism other than oxygen limitation.

Fundamental Flaws with the Fundamental Niche.

For more than 70 years, Hutchinson's concept of the fundamental niche has guided ecological research. Hutchinson envisioned the niche as a multidimensional hypervolume relating the fitness of an organism to relevant environmental factors. Here, we challenge the utility of the concept to modern ecologists, based on its inability to account for environmental variation and phenotypic plasticity. We have ample evidence that the frequency, duration, and sequence of abiotic stress influence the survivorship and performance of organisms. Recent work shows that organisms also respond to the spatial configuration of abiotic conditions. Spatiotemporal variation of the environment interacts with the genotype to generate a unique phenotype at each life stage. These dynamics cannot be captured adequately by a multidimensional hypervolume. Therefore, we recommend that ecologists abandon the niche as a tool for predicting the persistence of species and embrace mechanistic models of population growth that incorporate spatiotemporal dynamics.

The neuroscience of adaptive thermoregulation.

The nervous system acts as a biological thermostat by controlling behaviors that regulate the warming and cooling of animals. We review the structures responsible for thermoregulation in three model species: roundworms (Caenorhabditis elegans), flies (Drosophila melanogaster), and rats (Rattus novegicus). We then consider additional features of the nervous system required to explain adaptive plasticity of the set-point temperature and the precision of thermoregulation. Because animals use resources such as energy, water, and oxygen to thermoregulate, the nervous system monitors the abundance of these resources and adjusts the strategy of thermoregulation accordingly. Starvation, dehydration, or hypoxemia alter the activity of temperature-sensitive neurons in the pre-optic area of the hypothalamus. Other regions of the brain work in conjunction with the hypothalamus to promote adaptive plasticity of thermoregulation. For example, the amygdala likely inhibits neurons of the pre-optic area, overriding thermoregulation when a risk of predation or a threat of aggression exists. Moreover, the hippocampus enables an animal to remember microhabitats that enable safe and effective thermoregulation. In ectothermic animals, such as C. elegans and D. melanogaster, the nervous system can alter set-point temperatures as the environmental temperatures change. To build on this knowledge, neuroscientists can use experimental evolution to study adaptation of neural phenotypes in controlled thermal environments. A microevolutionary perspective would leverage our understanding of ecological processes to predict the origin and maintenance of neural phenotypes by natural selection.

Oxygen supply did not affect how lizards responded to thermal stress.

Zoologists rely on mechanistic niche models of behavioral thermoregulation to understand how animals respond to climate change. These models predict that species will need to disperse to higher altitudes to persist in a warmer world. However, thermal stress and, thus, thermoregulatory behavior may depend on atmospheric oxygen as well as environmental temperatures. Severe hypoxia causes animals to prefer lower body temperatures, which could be interpreted as evidence that oxygen supply limits heat tolerance. Such a constraint could prevent animals from successfully dispersing to high elevations during climate change. Still, an effect of oxygen supply on preferred body temperature has only been observed when oxygen concentrations fall far below levels experienced in nature. To see whether animals perceive greater thermal stress at an ecologically relevant level of hypoxia, we studied the thermoregulatory behavior of lizards (Sceloporus tristichus) exposed to oxygen concentrations of 13% and 21% (equivalent to PO2 at 4000 m and 0 m, respectively). In addition, we exposed lizards to 29% oxygen to see whether they would accept a higher body temperature at hyperoxia than at normoxia. At each oxygen level, we measured a behavioral response to heat stress known as the voluntary thermal maximum: the temperature at which a warming animal sought a cool refuge. Oxygen concentration had no discernable effect on the voluntary thermal maximum, suggesting that lizards experience thermal stress similarly at all 3 levels of oxygen (13%, 12% and 29%). Future research should focus on thermoregulatory behaviors under ecologically relevant levels of hypoxia.

Supply and demand: How does variation in atmospheric oxygen during development affect insect tracheal and mitochondrial networks?

Atmospheric oxygen is one of the most important atmospheric component for all terrestrial organisms. Variation in atmospheric oxygen has wide ranging effects on animal physiology, development, and evolution. This variation in oxygen has the potential to affect both respiratory systems (the supply side) and mitochondrial networks (the demand side) in animals. Insect respiratory systems supplying oxygen to tissues in the gas phase through blind ended tracheal systems are particularly susceptible to this variation. While the large conducting tracheae have previously been shown to respond developmentally to changes in rearing oxygen, the effect of oxygen on the tracheolar network has been relatively unexplored, especially in adult insects. Similarly, mitochondrial networks that meet energy demand in insects and other animals are dynamic and their enzyme activities have been shown to vary in the presence of oxygen. These two systems together should be under selective pressure to meet the aerobic metabolic requirements of insects. To test this hypothesis, we reared Mito-YFP Drosophila under three different oxygen concentrations hypoxia (12%), normoxia (21%), and hyperoxia (31%) and imaged their tracheolar and mitochondrial networks within their flight muscle using confocal microscopy. In terms of oxygen supply, hypoxia increased mean (mid-length) tracheolar diameters, tracheolar tip diameters, the number of tracheoles per main branch and affected tracheal branching patterns, while the opposite was observed in hyperoxia. In terms of oxygen demand, hypoxia increased mitochondrial investment and mitochondrial to tracheolar volume ratios; while the opposite was observed in hyperoxia. Generally, hypoxia had a stronger effect on both systems than hyperoxia. These results show that insects are capable of developmentally changing investment in both their supply and demand networks to increase overall fitness.

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.

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.

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.

Impaired climbing and flight behaviour in Drosophila melanogaster following carbon dioxide anaesthesia.

Laboratories that study Drosophila melanogaster or other insects commonly use carbon dioxide (CO2) anaesthesia for sorting or other work. Unfortunately, the use of CO2 has potential unwanted physiological effects, including altered respiratory and muscle physiology, which impact motor function behaviours. The effects of CO2 at different levels and exposure times were examined on the subsequent recovery of motor function as assessed by climbing and flight assays. With as little as a five minute exposure to 100% CO2, D. melanogaster exhibited climbing deficits up to 24 hours after exposure. Any exposure length over five minutes produced climbing deficits that lasted for days. Flight behaviour was also impaired following CO2 exposure. Overall, there was a positive correlation between CO2 exposure length and recovery time for both behaviours. Furthermore, exposure to as little as 65% CO2 affected the motor capability of D. melanogaster. These negative effects are due to both a CO2-specific mechanism and an anoxic effect. These results indicate a heretofore unconsidered impact of CO2 anaesthesia on subsequent behavioural tests revealing the importance of monitoring and accounting for CO2 exposure when performing physiological or behavioural studies in insects.

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.

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.

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.

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.