Subunit rotation in F0F1-ATP synthases as a means of coupling proton transport through F0 to the binding changes in F1

The rotation of an asymmetric core of subunits in F₀F₁-ATP synthases has been proposed as a means of coupling the exergonic transport of protons through F₀ to the endergonic conformational changes in F₁ required for substrate binding and product release. Here we review earlier evidence both for and against subunit rotation and then discuss our most recent studies using reversible intersubunit disulfide cross-links to test for rotation. We conclude that the subunit of F₁ rotates relative to the surrounding catalytic subunits during catalytic turnover by both soluble F₁ and membrane-bound F₀F₁. Furthermore, the inhibition of this rotation by the modification of F₀ with DCCD suggests that rotation in F₁ is obligatorily coupled to rotation in F₀ as an integral part of the coupling mechanism.     

玉米、花生及其间作茬口与施磷对冬小麦光合特性及产量的影响机制

该试验在玉米单作茬口、玉米-花生间作茬口(间作茬口)、花生单作茬口共3种茬口,以及0 kg P_2O_5·hm~(-2)(P_0)和180 kg P_2O_5·hm~(-2)(P_1) 2个磷水平下,研究了间作茬口与施磷对冬小麦分蘖、叶面积指数(LAI)、干物质积累、光合特性及产量的影响机制,为玉米花生间作与小麦-玉米复种轮作提供理论依据。结果表明:(1)间作茬口较玉米茬口显著提高了冬小麦有效分蘖数、LAI、净光合速率和干物质积累量,并提高了冬小麦旗叶的SPAD值、CO_2饱和点、光饱和点及最大净光合速率(P_(nmax))、表观量子效率(AQY)、羧化效率(CE)、最大羧化速率(V_(cmax))、最大RUBP再生的电子传递速率(J_(max))和最大磷酸丙糖利用速率(V_(TPU)),且CE、V_(cmax)、V_(TPU)的增幅均达到显著水平(P0.05),有效改善了冬小麦产量构成,显著提高籽粒产量(P0.05)。(2)间作茬口较花生茬口提高了冬小麦乳熟期的P_(nmax)、AQY、CE,增加了穗粒数和粒重,提高了产量。(3)与不施磷相比,施磷180 kg P_2O_5·hm~(-2)显著促进间作茬口冬小麦生长,显著提高冬小麦旗叶的SPAD值、P_(nmax)、AQY、CE、V_(cmax)、J_(max)、V_(TPU)和籽粒产量(P0.05)。研究发现,间作茬口较玉米茬口能有效增强冬小麦旗叶表观量子效率和CO_2羧化能力,显著提高小麦花后光合能力,促进冬小麦生长,从而增加穗粒数、粒重和籽粒产量,且间作茬口结合施磷180 kg P_2O_5·hm~(-2)效果更好。     

Effects of Forest Management on Total Biomass Production and CO<Subscript>2</Subscript> Emissions from use of Energy Biomass of Norway Spruce and Scots Pine

The aim of this study was to analyze the effects of forest management on the total biomass production (t ha^-1a^-1) and CO₂ emissions (kg CO₂ MWh^-1) from use of energy biomass of Norway spruce and Scots pine grown on a medium fertile site. In this context, the growth of both species was simulated using an ecosystem model (SIMA) under different management regimes, including various thinning and fertilization treatments over rotation lengths from 40 to 120 years in different pre-commercial stand densities. A Life Cycle Analysis/Emission calculation tool was employed to assess the CO₂ emissions per unit of energy from the use of biomass in energy production. Furthermore, the overall balance between the CO₂ uptake and emission (carbon balance) was studied, and the carbon neutrality (CN) factor was calculated to assess environmental effects of the use of biomass in energy production; i.e., how much CO₂ would be emitted per unit of energy when considering direct and indirect emissions from forest ecosystem and energy production. In general, the total annual biomass production for both species was highest when management with fertilization and high pre-commercial stand density (4000–6000 trees ha^-1) was used. In the case of Norway spruce, the highest annual biomass production was obtained with a rotation length of 80–100 years, while for Scots pine a rotation length of 40–60 years gave the highest annual production. In general, the CO₂ emissions decreased along with an increasing rotation length. The reduction was especially large if the rotation length was increased from 40 years to 60 years. Scots pine produced remarkably smaller net CO₂ emissions per year (on average 29%) than Norway spruce over all different densities and rotation lengths. The value of the CN factor was highest if a rotation of 100 years was used for Norway spruce stands and a rotation of 120 years for Scots pine. The CO₂ emission per energy unit was substantially less than that from the use of coal, which was used as reference to assess environmental effects of the use of biomass in energy production. The use of higher density of pre-commercial stand than that currently recommended in the Finnish forestry, together with timely thinning and fertilization, could increase the total biomass production, but also simultaneously decrease the net CO₂ emissions from the use of energy wood.     

The mechanism of rotating proton pumping ATPases

Two proton pumps, the F-ATPase (ATP synthase, F_oF₁) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F₁ or V₁) and a membrane intrinsic (F_o or V_o) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F₁ sector was found to rotate at surprisingly high speeds (> 400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F₁-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.     

Effect of external torque on the ATP-driven rotation of F1-ATPase

F₁-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F₀ to synthesize ATP in vivo. Therefore elucidation of the behavior of F₁ under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F₁ rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 μM), 120° stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.     

Elastic rotation of Escherichia coli FOF1 having ε subunit fused with cytochrome b562 or flavodoxin reductase

Intra-molecular rotation of F_OF₁ ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of F_OF₁ with the ε subunit connected to a globular protein [cytochrome b₅₆₂ (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction.     

Torque Generation Mechanism of F1-ATPase upon NTP Binding

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F₁-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10³ and 10⁶ times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F₁ to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F₁ exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F₁ with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.     

Protoplast rotation in a rotating electric field: The influence of cold acclimation

Summary An experimental and theoretical investigation has been made of the rotation of protoplasts ofSecale cereale L. (cv Puma) in a rotating electric field for the purpose of determining the electrical properties of the protoplast plasma membrane. The dependence of the protoplast rotation rate on: (1) the rotation rate of the applied electric field; (2) the electrical conductivity of the external medium; and (3) cold acclimation or lack thereof were determined. A theoretical analysis of the rotation rate of polarizable spherical cells in a rotating electric field leads to a qualitatively similar formula to that of Arnold and Zimmermann (Z. Naturforsch. 37:908–915, 1982), but it differs from this earlier work by a large numerical factor (180). Detailed comparisons of the observed protoplast rotation rates with the new theory show generally good agreement. The protoplast rotation measurements allow a noninvasive determination of the specific plasma membrane capacitance,c _m. The average value found in the present experiments isc _m=(0.56±0.08)×10^–2 F/m². Within the experimental errors, thec _m values are the same for cold-acclimated and noncold-acclimated protoplasts. Determination of plasma membrane resistance from protoplast rotation measurements does not appear feasible because of the high values of the specific resistance.     

Molecular basis of the protonophoric and uncoupling activities of the potent uncoupler SF-6847 ((3,5-di-tert-butyl-4-hydroxybenzylidene)malononitrile) and derivatives. Regulation of their electronic structures by restricted intramolecular rotation

We reported recently (Yoshikawa, K. and Terada, H. (1982) J. Am. Chem. Soc. 104, 7644–7646) that the potent uncoupler of oxidative phosphorylation SF-6847 ((3,5-di-tert-butyl-4-hydroxybenzylidene)malononitrile) shows unique intramolecular restricted rotation of the malononitrile moiety. In this study, values for the activation energy E_a of the restricted rotation of SF-6847 derivatives with the same alkyl chain R in both ortho positions of the phenolic hydroxyl group were determined from the temperature-dependent change in the¹H-NMR signals of their aromatic protons. The E_a values of the neutral forms of these derivatives were found to be the same irrespective of R, but those of the anionic forms increased with increase in the alkyl chain length of R. It was found that the restricted rotation of the malononitrile moiety regulates its electron-withdrawing ability in such a way as to keep the acid dissociability of these derivatives similar, overcoming the effect of steric hindrance by R. The protonophoric activity of these derivatives, in a phospholipid bilayer membrane and their uncoupling activity in rat-liver mitochondria were both found to depend on E_a of their anionic forms. The stability of the uncoupler anions regulated by the restricted rotation of the malononitrile group in a nonpolar membrane environment was found to be important for exhibition of these activities. The hydrophobicity of the anionic forms of these derivatives was suggested also to be affected by the intramolecular rotation.     

Torque Generation of Enterococcus hirae V-ATPase

V-ATPase (V_oV₁) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V_oV₁ for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V_oV₁ (EhV_oV₁) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV_oV₁ exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV_oV₁ only showed the “clear” state without apparent backward steps, whereas EhV₁ showed two states, “clear” and “unclear.” Furthermore, EhV_oV₁ showed slower rotation than EhV₁ without the three distinct pauses separated by 120° that were observed in EhV₁. When using a large probe, EhV_oV₁ showed faster rotation than EhV₁, and the torque of EhV_oV₁ estimated from the continuous rotation was nearly double that of EhV₁. On the other hand, stepping torque of EhV₁ in the clear state was comparable with that of EhV_oV₁. These results indicate that rotor-stator interactions of the V_o moiety and/or sodium ion transport limit the rotation driven by the V₁ moiety, and the rotor-stator interactions in EhV_oV₁ are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV₁. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV_oV₁.     

Torque Generation Mechanism of F1-ATPase upon NTP Binding

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F₁-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 10³ and 10⁶ times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F₁ to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F₁ exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F₁ with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity.     

31P nmr spin-lattice relaxation studies of deoxyoligonucleotides

Temperature-dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin-lattice relaxation times (T₁), and ³¹P-{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′-5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double- and single-stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10^?10 sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double-helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P-O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ? 10^?9 sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10^?10 sec, even for dinucleotides.     

Managing Carbon Sinks in Rubber (Hevea brasilensis) Plantation by Changing Rotation length in SW China

Extension of the rotation length in forest management has been highlighted in Article 3.4 of the Kyoto Protocol to help the countries in their commitments for reduction in greenhouse gas emissions. CO₂FIX Model Ver.3.2 was used to examine the dynamics of carbon stocks (C stocks) in a rubber plantation in South Western China with the changing rotation lengths. To estimate the efficiency of increasing the rotation length as an Article 3.4 activity, study predicted that the rubber production and C stocks of the ecosystem increased with the increasing rotation (25, 30, 35, 40 and 45 years). While comparing the pace of growth both in economical (rubber production) and ecological (C stocks) terms in each rotation, 40 years rotation length showed maximum production and C stocks. After elongation of 40 year rotation to four consecutive cycles, it was concluded that the total C stocks of the ecosystem were 186.65 Mg ha^-1. The longer rotation lengths showed comparatively increased C stocks in below ground C stock after consecutive four rotations. The pace of C input (Mg C ha^-1yr^-1) and rubber production indicated that 40years rotation is best suited for rubber plantation. The study has developed carbon mitigation based on four rotation scenarios. The possible stimulated increase in C stocks of the entire ecosystem after consecutive long rotations indicated that the emphasis must be paid on deciding the rotation of rubber plantation in SW China for reporting under article 3.4 of the Kyoto Protocol.     

Irregular activity oscillations of a rotary molecular motor: A simple kinetic model of F<Subscript>1</Subscript>-ATPase

F₁-ATPase is a catalytic part of the F₁F_o-ATP synthase molecular motor. The cooperative hydrolysis of ATP at three catalytic sites of F₁-ATPase is accompanied by the rotation of the central γ-subunit inside a cylinder formed by three α-subunits and three β-subunits. Experimental works of different authors have shown that the γ-subunit rotates with irregular dwells. A simple kinetic model suggested in this article provides an explanation as to why dwells occur during the rotation of F₁-ATPase. According to this model, rotation dwells happen as a result of deterministic chaos, which in turn occurs at rate constants that are close to those demonstrated experimentally. The time duration of dwells in the model is in agreement with that observed experimentally. Our model explains the known irregular occupancy of catalytic sites of F₁-ATPase by nucleotides.     

Impacts of Intensive Management and Landscape Structure on Timber and Energy Wood Production and net CO2 Emissions from Energy Wood Use of Norway Spruce

The aim of this study was to analyze the effects of intensive management and forest landscape structure (in terms of age class distribution) on timber and energy wood production (m³?ha^?1), net present value (NPV, ? ha^?1) with implications on net CO₂ emissions (kg CO₂ MWh^?1 per energy unit) from energy wood use of Norway spruce grown on medium to fertile sites. This study employed simulations using a forest ecosystem model and the Emission Calculation Tool, considering in its analyses: timber (saw logs, pulp) and energy wood (small-sized stem wood and/or logging residuals for top part of stem, branches, and needles) from the first thinning and harvesting residuals and stumps from the final felling. At the stand level, both fertilization and high pre-commercial stand density clearly increased timber production and the amount of energy wood. Short rotation length (40 and 60?years) outputted, on average, the highest annual stem wood production (most fertile and medium fertile sites), the 60?year rotation also outputted the highest average annual net present value (NPV with interest rates of 1?C4%). On the other hand, even longer rotation lengths, up to 80 and 100?years, were needed to output the lowest net CO₂ emissions per year in energy wood use. At the landscape level, the largest productivity (both for timber and energy wood) was obtained using rotation lengths of 60 and 80?years with an initial forest landscape structure dominated by older mature stands (a right-skewed age-class distribution). If the rotation length was 120?years, the initial forest landscape dominated by young stands (a left-skewed age-class distribution) provided the highest productivity. However, the NPV with interest rate of 2% was, on average, the highest with a right-skewed distribution regardless of the rotation length. If the rotation length was 120?years, normal age class distribution provided, on average, the highest NPV. On the other hand, the lowest emissions (kg CO₂ MWh^?1a^?1) were obtained with the left-skewed age-class distribution using the rotation lengths of 60 and 80?years, and with the normal age-class distribution using the rotation length of 120?years. Altogether, the management regimes integrating both timber and energy wood production and using fertilization provided, on average, the lowest emissions over all management alternatives considered.     

On chemomechanical coupling of the F1-ATPase molecular motor

F₁-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F₁-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F₁-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well.     

Free Atmospheric CO2 Enrichment (FACE) Increased Labile and Total Carbon in the Mineral Soil of a Short Rotation Poplar Plantation

The global net terrestrial carbon sink was estimated to range between 0.5 and 0.7 Pg C y⁻¹ for the early 1990s. FACE (free atmospheric CO₂ enrichment) studies conducted at the whole-tree and community scale indicate that there is a marked increase of primary production, mainly allocated into below-ground biomass. The enhanced carbon transfer to the root system may result in enhanced rhizodeposition and subsequent transfer to soil C pools. During the first rotation of the POP/EuroFACE experiment in a short-rotation Poplar plantation, total soil C content increased more under ambient CO₂ treatment than under FACE, while under FACE more new C was incorporated than under ambient CO₂. These unexpected and opposite effects may have been caused by a priming effect, where priming effect is defined as the stimulation of SOM decomposition caused by the addition of labile substrates. In order to gain insight into these processes affecting SOM decomposition, we obtained the labile, refractory and stable pools of soil C and N by chemical fractionation (acid hydrolysis) and measured rates of N-mineralization. Results of the first 2 years of the second rotation show a larger increase of total soil C% under FACE than under ambient CO₂. In contrast to the first rotation, total C% is now increasing faster under FACE than under ambient CO₂. Based on these observations we infer that the priming effect ceased during the second rotation. FACE treatment increased the labile C fraction at 0–10 cm depth, which is in agreement with the larger input of plant litter and root exudates under FACE. N-mineralization rates were not affected by FACE. We infer that the system switched from a state where extra labile C and sufficient N-availability (due to the former agricultural use of the soil) caused a priming effect (first rotation), to a state where extra C input is accumulating due to limited N-availability (second rotation). Our results on N-mineralization (second rotation) are in agreement with observations made at three forest FACE sites (Duke Forest, Oak Ridge, and Rhinelander), but our finding of increasing mineral soil C content contrasted with results at the Duke Forest where no significant increase in C content of the mineral soil occurred. However, the FACE induced increase in total C content occurred within the fraction with the shortest turnover time, i.e. the labile fraction. The refractory and stable fractions were not affected. The question remains whether the currently observed larger increase of total soil C and the increase of labile C under FACE will eventually result in long-term C storage in refractory and stable organic matter fractions.     

Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage

Responses of freshwater organisms to environmental oxygen tensions (PO₂) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO₂ must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO₂s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO₂s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (“stir-bar” theory of embryonic rotational behaviour).