Cyanobacterial NDH-1 complexes: novel insights and remaining puzzles

Cyanobacterial NDH-1 complexes belong to a family of energy converting NAD(P)H:Quinone oxidoreductases that includes bacterial type-I NADH dehydrogenase and mitochondrial Complex I. Several distinct NDH-1 complexes may coexist in cyanobacterial cells and thus be responsible for a variety of functions including respiration, cyclic electron flow around PSI and CO(2) uptake. The present review is focused on specific features that allow to regard the cyanobacterial NDH-1 complexes, together with NDH complexes from chloroplasts, as a separate sub-class of the Complex I family of enzymes. Here, we summarize our current knowledge about structure of functionally different NDH-1 complexes in cyanobacteria and consider implications for a functional mechanism. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Cyanobacterial NADPH dehydrogenase complexes

Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO₂ uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO₂ uptake. Other than CO₂ uptake, chloroplastic NDH-1 complex has a similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described for comparison.     

Expression and activity of type 1 NAD(P)H dehydrogenase at different growth phases of the cyanobacterium, Synechocystis PCC 6803

The expression and activity of type 1 NAD(P)H dehydrogenase (NDH-1) were investigated in Synechocystis PCC 6803 cells during different growth phases (i.e. lag, logarithmic, stationary and decline phases). The relative amount of NDH-1, estimated by Western blot analysis using antibodies against NdhH, NdhI and NdhK, increased more than two-fold during growth from the lag to the logarithmic phase and then decreased after the logarithmic phase to reach lowest levels after 15 days (decline phase). The activity of light-dependent NADPH oxidation and cyclic electron flow around photosystem I (PSI) changed nearly in parallel with the amount of NdhH, NdhI and NdhK in cells across the growth phases. In contrast, the activity of photosynthetic O₂ evolution and respiratory O₂ uptake was not significantly different across phases of growth; the fluctuation of the activity at different phases was within 40%. These results suggested that the activity of light-dependent NADPH oxidation and PSI-cyclic electron flow are restricted by the amount of NDH-1 and that other factor(s) are limiting the rates of photosynthesis and respiration.     

几种蓝藻光合NAD（P）H脱氢酶复合体研究的新进展

蓝藻NAD（P）H脱氢酶（NDH-1）是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。就几种蓝藻NDH-1复合体的鉴定、结构、生理功能等研究的新进展进行了综述与分析,并对今后NDH-1复合体的研究作了展望。     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Adaptation to shade of the light-harvesting apparatus in Silene dioica

Abstract. The physiological characteristics and photo-system composition of the photosynthetic apparatus of Silene dioica , a woodland plant, grown in sun and natural shade are examined. As expected, shade leaves exhibited lower chlorophyll a/b ratios, light saturated rates of CO₂ assimilation (A_sat), dark respiration (R_d,) and light compensation points ( Г ), with both sun and shade leaves having similar absorptances and quantum yields of CO₂ assimilation (φ). Shade leaves were able to utilize far-red light for electron transport and carbon assimilation and reach the compensation point. Sun leaves in far-red light had a rate of carbon assimilation equivalent to their dark respiration rate. Chlorophyll fluorescence kinetics from leaves at 77 K together with analyses of thylakoid contents of photosystems (PS) I and II and the light-harvesting cholorphyll a/b protein complex associated with PSII (LHCII) demonstrated that the antenna size of PSII was similar in thylakoids of sun and shade leaves, but shade leaves contained ca. 20% more PSII and ca. 12% less PSI complexes. The increased PSII/PSI ratio in shade leaves accounted for their ability to achieve the compensation point in far-red light. An important feature of photosynethic shade adaptation in S. dioica is an increase in the PSII/PSI ratio and not an increase in the antenna size of PSII. The adaptive response of sun leaves when placed in a shade environment was rapid and had a half-time of ca. 18h.     

Changes in leaf nitrogen and carbohydrates underlie temperature and CO2 acclimation of dark respiration in five boreal tree species

We tested the hypothesis that acclimation of foliar dark respiration to CO₂ concentration and temperature is associated with adjustments in leaf structure and chemistry. Populus tremuloides Michx. , Betula papyrifera Marsh. , Larix laricina (Du Roi) K. Koch , Pinus banksiana Lamb., and Picea mariana (Mill.) B.S.P. were grown from seed in combined CO₂ (370 or 580 μ mol mol^–1) and temperature treatments (18/12, 24/18, or 30/24 °C). Temperature and CO₂ effects were predominately independent. Specific respiration rates partially acclimated to warmer thermal environments through downward adjustment in the intercept, but not Q ₁₀ of the temperature–response functions. Temperature acclimation of respiration was larger for conifers than broad-leaved species and was associated with pronounced reductions in leaf nitrogen concentrations in conifers at higher growth temperatures. Short-term increases in CO₂ concentration did not inhibit respiration. Growth in the elevated CO₂ concentration reduced leaf nitrogen and increased non-structural carbohydrate concentrations. However, for a given nitrogen concentration, respiration was higher in leaves grown in the elevated CO₂ concentration, as rates increased with increasing carbohydrates. Across species and treatments, respiration rates were a function of both leaf nitrogen and carbohydrate concentrations ( R ² = 0·71, P < 0·0001). Long-term acclimation of foliar dark respiration to temperature and CO₂ concentration is largely associated with changes in nitrogen and carbohydrate concentrations.     

Influence of soil O2 and CO2 on root respiration for Agave deserti

Respiration measured as CO₂ efflux was determined at various soil O₂ and CO₂ concentrations for individual, attached roots of a succulent perennial from the Sonoran Desert, Agave deserti Engelm. The respiration rate increased with increasing O₂ concentration up to about 16% O₂ for established roots and 5% O₂ for rain roots (fine branch roots on established roots induced by wetting of the soil) and then remained fairly constant up to 21% O₂. When O₂ was decreased from 21 to 0%, the respiration rates were similar to those obtained with increasing O₂ concentration. The CO₂ concentration in the root zone, which for the shallow-rooted A. deserti in the field was about 1 000 μl l^-1, did not affect root respiration at concentrations up to 2 000 μl l^-1, but higher concentrations reduced it, respiration being abolished at 20 000 μl l^-1 (2%) CO₂ for both established and rain roots. Upon lowering CO₂ to 1 000 μl l^-1 after exposure to concentrations up to 10000 μl l^-1 CO₂, inhibition of respiration was reversible. Uptake of the vital stain neutral red by root cortical cells was reduced to zero, indicating cell death, in about 4 h at 2% CO₂, substantiating the detrimental effects of high soil CO₂ concentrations on roots of A. deserti . This CO₂ response may explain why roots of desert succulents tend to occur in porous, well-aerated soils.     

Why do fast- and slow-growing grass species differ so little in their rate of root respiration,considering the large differences in rate of growth and ion uptake?

Herbaceous plants grown with free access to nutrients exhibit inherent differences in maximum relative growth rate (RGR) and rate of nutrient uptake. Measured rates of root respiration are higher in fast-growing species than in slow-growing ones. Fast-growing herbaceous species, however, exhibit lower rates of respiration than would be expected from their high rates of growth and nitrate uptake. We investigated why the difference in root O₂ uptake between fast- and slow-growing species is relatively small. Inhibition of respiration by the build-up of CO₂ in closed cuvettes, diurnal variation in respiration rates or an increasing ratio of respiratory CO₂ release to O₂ uptake (RQ) with increasing RGR failed to explain the relatively low root respiration rates in fast-growing grasses. Furthermore, differences in alternative pathway activity can at most only partly explain why the difference in root respiration between fast- and slow-growing grasses is relatively small. Although specific respiratory costs for maintenance of biomass are slightly higher in the fast-growing Dactylis glomerata L. than those in the slow-growing Festuca ovina L., they account for 50% of total root respiration in both species. The specific respiratory costs for ion uptake in the fast-growing grass are one-third of those in the slow-growing grass [0·41 versus 1·22 mol O₂ mol (NO₃^–)^–1]. We conclude that this is the major cause of the relatively low rates of root respiration in fast-growing grasses.     

Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases

We present, for the first time, the oxygen response kinetics of mitochondrial respiration measured in intact leaves (sunflower and aspen). Low O₂ concentrations in N₂ (9–1500 ppm) were preset in a flow-through gas exchange measurement system, and the decrease in O₂ concentration and the increase in CO₂ concentration as result of leaf respiration were measured by a zirconium cell O₂ analyser and infrared-absorption CO₂ analyser, respectively. The low O₂ concentrations little influenced the rate of CO₂ evolution during the 60-s exposure. The initial slope of the O₂ uptake curve on the dissolved O₂ concentration basis was relatively constant in leaves of a single species, 1.5 mm s⁻¹ in sunflower and 1.8 mm s⁻¹ in aspen. The apparent K _0.5(O₂) values ranged from 0.33 to 0.67 μ M in sunflower and from 0.33 to 1.1 μ M in aspen, mainly because of the variation of the maximum rate, V _max (leaf temperature 22°C). The initial slope of the O₂ response of respiration characterizes the catalytic efficiency of terminal oxidases, an important parameter of the respiratory machinery in leaves. The plateau of the response characterizes the activity of the mitochondrial electron transport chain and is subject to regulations in accordance with the necessity for ATP production. The relatively low oxygen conductivity of terminal oxidases means that in leaves, less than 10% of the photosynthetic oxygen can be reassimilated by mitochondria.     

CaCO3 nucleation by cyanobacteria: laboratory evidence for a passive, surface-induced mechanism

Calcite nucleation on the surface of cyanobacteria of the Synechococcus leopoliensis strain PCC 7942 was investigated to assess the influence of photosynthetic uptake of inorganic carbon and active ion exchange processes across the cell membrane on the nucleation and precipitation mechanisms. We performed long-term precipitation experiments at a constant CO₂ level in ambient air by adding suspensions of previously washed cyanobacteria to solutions of NaHCO₃/CaCl₂ which were supersaturated with respect to calcite. Induction times between 4 and 110 h were measured over a range of saturation states, Ω, between 8 and 4. The kinetics of CaCO₃ nucleation was compared between experiments: (i) with ongoing photosynthesis, (ii) with cells metabolizing but not undergoing photosynthetic uptake of inorganic carbon and (iii) in darkness without photosynthesis. No significant differences were observed between the three treatments. The results reveal that under low nutrient concentrations and permanent CO₂ supply, photosynthetic uptake of inorganic carbon predominantly uses CO₂ and consequently does not directly influence the nucleation process of CaCO₃ at the surface of S. leopoliensis. Furthermore, ion exchange processes did not affect the kinetics, indicating a passive nucleation process wherein the cell surface or extracellular polymers provided preferential sites for mineral nucleation. The catalyzing effect of the cyanobacteria on calcite nucleation was equivalent to a ∼18% reduction in the specific interfacial free energy of the calcite nuclei. This result and the ubiquitous abundance of cyanobacteria suggest that this process may have an impact on local and global carbon cycling.     

NdhM Subunit Is Required for the Stability and the Function of NAD(P)H Dehydrogenase Complexes Involved in CO2 Uptake in Synechocystis sp. Strain PCC 6803

The cyanobacterial type I NAD(P)H dehydrogenase (NDH-1) complexes play a crucial role in a variety of bioenergetic reactions such as respiration, CO₂ uptake, and cyclic electron transport around photosystem I. Two types of NDH-1 complexes, NDH-1MS and NDH-1MS′, are involved in the CO₂ uptake system. However, the composition and function of the complexes still remain largely unknown. Here, we found that deletion of ndhM caused inactivation of NDH-1-dependent cyclic electron transport around photosystem I and abolishment of CO₂ uptake, resulting in a lethal phenotype under air CO₂ condition. The mutation of NdhM abolished the accumulation of the hydrophilic subunits of the NDH-1, such as NdhH, NdhI, NdhJ, and NdhK, in the thylakoid membrane, resulting in disassembly of NDH-1MS and NDH-1MS′ as well as NDH-1L. In contrast, the accumulation of the hydrophobic subunits was not affected in the absence of NdhM. In the cytoplasm, the NDH-1 subcomplex assembly intermediates including NdhH and NdhK were seriously affected in the ΔndhM mutant but not in the NdhI-deleted mutant ΔndhI. In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS′ involved in CO₂ uptake in Synechocystis sp. strain PCC 6803.     

Respiration in a future, higher-CO2 world

Abstract. Apart from its impact on global warming, the annually increasing atmospheric [CO₂] is of interest to plant scientists primarily because of its direct influence on photosynthesis and photorespiration in C₃ species. But in addition, 'dark' respiration, another major component of the carbon budget of higher plants, may be affected by a change in [CO₂] independent of an increase in temperature. Literature pertaining to an impact of [CO₂] on respiration rate is reviewed. With an increase in [CO₂], respiration rate is increased in some cases, but decreased in others. The effects of [CO₂] on respiration rate may be direct or indirect. Mechanisms responsible for various observations are proposed. These proposed mechanisms relate to changes in: (1) levels of nonstructural carbohydrates, (2) growth rate and structural phytomass accumulation, (3) composition of phytomass, (4) direct chemical interactions between CO₂ and respiratory enzymes, (5) direct chemical interactions between CO₂ and other cellular components, (6) dark CO₂ fixation rate, and (7) ethylene biosynthesis rate. Because a range-of (possibly interactive) effects exist, and present knowledge is limited, the impact of future [CO₂] on respiration rate cannot be predicted. Theoretical considerations and types of experiments that can lead to an increase in the understanding of this issue are outlined.     

The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO₂ concentration, and not detectable in the NDH-1MS and NDH-1MS'' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.     

Mitigation of adverse effects of rising CO2 on a planktonic herbivore by mixed algal diets

Putative future increase in atmospheric CO₂ is expected to adversely affect herbivore growth due to decrease in contents of key nutrients such as nitrogen and phosphorus (P) relative to carbon in primary producers including plant and algal species. However, as many herbivores are polyphagous and as the response of primary producers to elevated CO₂ is highly species-specific, effects of elevated CO₂ on herbivore growth may differ between feeding conditions with monospecific and multiproducer diets. To examine this possibility, we performed CO₂ manipulation experiments under a P-limited condition with a planktonic herbivore, Daphnia , and three algal species, Scenedesmus obliquus (green algae), Cyclotella sp. (diatoms) and Synechococcus sp. (cyanobacteria). Semibatch cultures with single algal species (monocultures) and multiple algal species (mixed cultures) were grown at ambient (360 ppm) and high CO₂ levels (2000 ppm) that were within the natural range in lakes. Both in the mono- and mixed cultures, algal steady state abundance increased but algal P : C and N : C ratios decreased when they were grown at high CO₂. As expected, Daphnia fed monospecific algae cultured at high CO₂ had decreased growth rates despite increased algal abundance. However, when fed mixed algae cultured at high CO₂, especially consisting of diatoms and cyanobacteria or the three algal species, Daphnia maintained high growth rates despite lowered P and N contents relative to C in the algal diets. These results imply that algal diets composed of multiple species can mitigate the adverse effects of elevated CO₂ on herbivore performance, although the magnitude of this mitigation depends on the composition of algal species involved in the diets.     

Respiratory responses of higher plants to atmospheric CO2 enrichment

Although the respiratory response of native and agricultural plants to atmospheric CO₂ enrichment has been reported over the past 75 years, only recently have these effects emerged as prominent measures of plant and ecosystem response to the earth's changing climate. In this review we discuss this rapidly expanding field of study and propose that both increasing and decreasing rates of leaf and whole-plant respiration are likely to occur in response to rising CO₂ concentrations. While the stimulatory effects of CO₂ on respiration are consistent with our knowledge of leaf carbohydrate status and plant metabolism, we wish to emphasize the rather surprising short-term inhibition of leaf respiration by elevated CO₂ and the reported effects of long-term CO₂ exposure on growth and maintenance respiration. As is being found in many studies, it is easier to document the respiratory response of higher plants to elevated CO₂ than it is to assign a mechanistic basis for the observed effects. Despite this gap in our understanding of how respiration is affected by CO₂ enrichment, data are sufficient to suggest that changes in leaf and whole-plant respiration may be important considerations in the carbon dynamics of terrestrial ecosystems as global CO₂ continues to rise. Suggestions for future research that would enable these and other effects of CO₂ on respiration to be unravelled are presented.     

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat

The temperature dependence of C₃ photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO₂ on photosynthesis temperature response have been investigated in wheat ( Triticum aestivum L.) grown in controlled chambers with 370 or 700 μmol mol⁻¹ CO₂ from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO₂ slightly decreased the CO₂ compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V_cmax, although the latter effect was reversed at 15°C. With elevated CO₂, J_max decreased in the 15–25°C temperature range and increased at 30 and 35°C. The temperature response (activation energy) of V_cmax and J_max increased with growth in elevated CO₂. CO₂ enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO₂. We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO₂ on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.     

Carbon dioxide and ethylene interactions in tulip bulbs

The effect of CO₂ on ethylene-induced gummosis (secretion of polysaccharides), weight loss and respiration in tulip bulbs ( Tulipa gesneriana L.) was investigated. A pretreatment with 1-MCP prevented these ethylene-induced effects, indicating that ethylene action must have been directed via the ethylene receptor. Treatment with 0.3 Pa ethylene for 2 days caused gummosis on 50% of the total number of bulbs of cultivar Apeldoorn, known to be sensitive for gummosis. Addition of CO₂ (10 kPa) reduced the ethylene-induced gummosis to 18%. In a second experiment the influence of ethylene and CO₂ on respiration and FW loss of bulbs of the cultivar Leen van der Mark was studied. A range of ethylene partial pressures (0.003–0.3 Pa) was applied continuously for 29 days. Ethylene caused a transient peak in O₂ consumption rate during the first days after the start of application. The relation between O₂ consumption rate and ethylene partial pressure could be described by Michaelis-Menten kinetics. Respiratory peaks were reduced by CO₂. This inhibition by CO₂ could not totally be due to competition with ethylene at the receptor binding-site, as was indicated by the use of an O₂ consumption model. Pre-treatment of bulbs with 1-MCP and subsequent exposure to CO₂ showed that CO₂ could influence respiration irrespective of any interaction with ethylene. Ethylene and CO₂ both stimulated weight loss. The effect of combined treatments of ethylene and CO₂ on weight loss was at least as strong as the sum of the separate effects, which implies that competition between ethylene and CO₂ at the receptor binding-site was unlikely.     

Immunolocalization of the uptake hydrogenase in the marine cyanobacterium Lyngbya majuscula CCAP 1446/4 and two Nostoc strains

In N₂-fixing cyanobacteria, the reduction of N₂ to NH₃ is coupled with the production of molecular hydrogen, which is rapidly consumed by an uptake hydrogenase, an enzyme that is present in almost all diazotrophic cyanobacteria. The cellular and subcellular localization of the cyanobacterial uptake hydrogenase remains uncertain, and it is definitely strain dependent. Previous studies focused mainly on heterocystous cyanobacteria and used heterologous antisera. The present work represents the first effort to establish the subcellular localization of the uptake hydrogenase in a N₂-fixing filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4, using the first antiserum produced against a cyanobacterial uptake hydrogenase. The data obtained revealed higher specific labelling associated with the thylakoid membranes of L. majuscula , reinforcing the idea that the cyanobacterial uptake hydrogenase is indeed a membrane-bound protein. For comparative purposes, the localization of the uptake hydrogenase was also investigated in two distinct heterocystous cyanobacterial strains, and while in Nostoc sp. PCC 7120 the labelling was only observed in the heterocysts, in Nostoc punctiforme , the presence of uptake hydrogenase antigens was detected in both the vegetative cells and heterocysts, corresponding most probably to an inactive and an active form of the enzyme.     

Rhythms of passive and active ventilation, and circulation recorded in diapausing pupae of Mamestra brassicae using constant volume respirometry

Abstract.  The periodically occurring convective inflow of air into the tracheal system, or passive suction ventilation, together with the cyclic bursts of release of CO₂ and active ventilation, is recorded in diapausing pupae of Mamestra brassicae . A constant volume respirometer combined with an opto-cardiograph-actograph is used. In all pupae with a metabolic rate of 0.025–0.054 mL g⁻¹ h⁻¹, the bouts of almost imperceptible abdominal contractions are recorded during the bursts of carbon dioxide release and this mode of active ventilation is qualified as extracardiac haemocoelic pulsations. The pupae whose metabolic rate is 0.052–0.075 mL⁻¹ g⁻¹ h⁻¹ show more vigorous abdominal contractions. The results demonstrate that, in diapausing pupae, characterized with low metabolic rates, both passive suction ventilation, referred to also as passive suction inspiration, and active ventilation occurs. In approximately 50% of the pupae, each gas exchange microcycle during the interburst periods begins with a miniature PSI followed by a microburst of CO₂ release; in approximately 30% of the individuals, passive suction inspirations occur separately from CO₂ microbursts; in the remaining pupae, miniature ones without microbursts of CO₂ are recorded. A typical event is heartbeat reversion: in longer periods, the heart peristalses are directed forward (anterograde of heartbeat) and, in shorter periods, the heart peristalses are directed backward (retrograde of heartbeat). At 0 °C, the cyclic release of CO₂ and miniature passive suction inspirations during the interburst periods are preserved at lower frequencies but active ventilation is lost.