Cartesian Diver Microrespirometry Research Articles

Claes Hellerström and Cartesian diver microrespirometry

Cartesian diver microrespirometry was introduced by Claes Hellerström at the Department of Histology/Medical Cell Biology at Uppsala University, Sweden, to determine rates of oxygen consumption in islets of Langerhans. The theory behind this method is touched upon and the main findings described. Glucose-stimulated beta cell respiration significantly contributes to increased ATP generation, which is a prerequisite for stimulated insulin secretion and synthesis. This has had major implications for understanding the beta cell stimulus–secretion coupling.

The oxygen consumption rates of different life stages of the endoparasitic nematode

The oxygen consumption rates of different life stages of the endoparasitic nematode, Pratylenchus zeae (Nematoda: Tylenchida) during non- and post-anhydrobiosisPratylenchus zeae, widely distributed in tropical and subtropical regions, is an endoparasite in roots of maize and other crop plants. The nematode is attracted to plant roots by CO2 and root exudates and feeds primarily on cells of the root cortex, making channels and openings where the eggs are deposited, with the result that secondary infection occurs due to bacteria and fungi. Nothing is known about the respiration physiology of this nematode and how it manages to survive during dry seasons. To measure the oxygen consumption rate (VO2 ) of individual P. zeae (less than half a millimeter long), a special measuring technique namely Cartesian diver micro-respirometry was applied. The Cartesian divers were machined from Perspex, and proved to be more accurate to measure VO2 compared with heavier glass divers used in similar experiments on free living nematodes. An accuracy of better than one nanoliter of oxygen consumed per hour was achieved with a single P. zeae inside the diver. Cartesian diver micro-respirometry measurements are based in principle on the manometric changes that occur in a fl otation tube in a manometer set-up when oxygen is consumed by P. zeae and CO2 from the animal is chemically absorbed. VO2 was measured for eggs (length: < 0.05 mm), larvae (length: 0.36 mm) and adults (length: 0.47 mm) before induction to anhydrobiosis. P. zeae from infected maize roots were extracted and exposed aseptically to in vitro maize root cultures in a grow cabinet at 50 % to 60% relative humidity at 28 ºC using eggs, larvae and adults. VO2 was also measured for post-anhydrobiotic eggs, larvae and adults by taking 50 individuals, eggs and larvae from the culture and placing them in Petri-dishes with 1% agar/water to dry out for 11 days at 28 ºC and 50% relative humidity. The VO2 was measured after the anhydrobiotic eggs, larvae and adults were re-hydrated for 12 hours in a high humidity atmosphere. The average VO2 value found for ten consecutive measurements during a 50 minute period of one adult using the diver technique was 32.8 nanoliter per hour. The differences between the ten VO2 values were less than 3.5 %, an indication of the accuracy of the diver measurements. The average VO2 values from ten measurements per life stage, expressed in nanolitres per hour per life stage of the pre-anhydro-biotes (eggs: 7.96; larva: 6.13; adult: 26.04) were compared with those of post-anhydrobiotes 12 hours after anhydrobiosis. The average VO2 values of the post-anhydrobiotes for the three life stages (egg: 19.34; larva: 14.17; adult: 32.86) were statistically signifi cantly higher in comparison with the pre-anhydrobiotes. The reasons for the difference are that high concentrations of metabolites, probably in the form of trehalose, accumulate during the anhydrobiosis stage to be utilized during the post-anhydrobiotic revival period. The oxygen consumption rate was also expressed in nanolitres per hour per microgram adult nematode after applying the following equation taken from the literature: M = a2 x b/16 x 1000 where M = mass (µg) of adult nematode; a = largest body width (µm); b = body length (µm). Using this equation it was found that one gram P. zeae uses 503 times more oxygen compared with one gram mammal the size of a cow. This high specifi c oxygen consumption rate (MO2 ) is a direct indication of the large metabolic damage this endoparasitic nematode can have on the metabolic substrates provided by the roots of the various plant crops it parasitize.

Respiration rates and biovolumes of common benthic Foraminifera (Protozoa)

The respiration rates of five genera of benthic Foraminifera were determined by Cartesian diver microrespirometry. Across all genera studied, the rate averaged 11·3×10−3μl O2individual−1h−1at 10°C. Estimates of foraminiferal mean biovolumes ranged from 0·66 to 6·54×106μm3, giving an overall mean volume-specific respiration rate of 7·36 × 10−9μl O2h−1μm−3. For non-symbiont-bearing Foraminifera, in general, volume-specific rates (log μl O2h−1μm3) are best described by the equation, −0·98 × (log cell biovolume, μm3)−2·01. The respiration results show that these benthic Foraminifera respire some ten times more rapidly than naked amoebae of equivalent size. The combination of high respiratory rates and the often large standing stocks of Foraminifera encountered, suggests that these organisms may contribute significantly to total microbial benthic respiration.

Measurement of guard-cell respiration rates using Cartesian-diver technique

Dark respiration rates of guard-cell protoplasts of Commelina communis L. were measured over a temperature range (15-30° C) using a Cartesian-diver microrespirometry technique. Measurements were made using a few microliters of suspension medium containing between 400 and 3 700 protoplasts. Respiration rates were approximately linear for at least 1 h at all temperatures. Respiration rates increased rapidly between 20 and 25° C to relatively high levels (6.11·10(-6) μmol O2 h(-1) protoplast(-1)=1259 μmol O2 mg(-1) chlorophyll h(-1)=22.97 μmol O2 mg(-1) protein h(-1)) with no further increases above this temperature. Respiration rates were much lower in protoplasts 15-16 h old than in freshly prepared ones indicating considerable deterioration of their viability over this time period.

Respiratory metabolism of Cyclops bicuspidatus (sensu stricta) (Claus) (Copepoda: Cyclopoida) from Esthwaite Water, Cumbria.

The respiration rate of all stages of Cyclops bicuspidatus (s. str.) (Claus), a benthic copepod, from Esthwaite Water in the English Lake District, was determined at a range of field temperatures, 4° C-12° C, using cartesian diver microrespirometry. The population of C. bicuspidatus in Esthwaite Water was found to be adapted to low temperatures, with an optimum rate of metabolism at 8° C. Weight varied with temperature, in general the largest weights occurred at the lower temperatures. Adult males had higher rates of respiration than adult females, which were on average twice the size of males. Gravid and non-gravid females had similar levels of metabolism. Regression coefficients (b), derived from regressions of log oxygen consumption against log dry weight were low, ranging between 0.25-0.51 according to temperature.

Respiratory energy losses in the protozoan predator Didinium nasutum Müller (Ciliophora).

Respiration in Didinium nasutum, an active protozoan predator, was investigated in relation to cell weight at 10, 15, and 20° C by means of cartesian diver microrespirometry. Oxygen uptake increased progressively over the 10-20° C temperature range; a table of Q 10 related to weight is presented. Regression coefficients of log weight versus log oxygen uptake (b) were 0.96 at 10° C, 0.98 at 15° C and 1.00 at 20° C. D. nasutum was shown to expend very much higher levels of respiratory energy than a mainly sedentary carnivorous ciliate of comparable weight.

Respiratory Energy Losses in Podophrya fixa Muller in Relation to Temperature and Nutritional Status

SUMMARY: Respiration in well-fed and starved Podophrya fixa was measured by means of Cartesian diver microrespirometry at 5 °C intervals between 10 °C and 30 °C. At 10C encystment occurred. The optimum temperature for respiration was 25C where a rate of 41·3 × 10−6 μl h−1 per individual was recorded in well-fed animals. Mean cell size also reached a maximum at 25 °C in well-fed individuals. Both respiration rate and mean cell size decreased with starvation, as did the rate of metabolism. A table of daily respiratory energy losses in well-fed and starved Podophrya is presented. The results and their implications are discussed in relation to other protozoa.

Respiratory energy losses related to cell weight and temperature in ciliated protozoa.

Respiratory energy losses in five species of ciliated protozoa, Tetrahymena pyriformis Ehrenberg, Vorticella microstoma Ehrenberg, Paramecium aurelia Ehrenberg, Spirostomum teres Claparède and Lachmann and Frontonia leucas Ehrenberg, were investigated at 8.5° C, 15° C and 20° C using Cartesian diver microrespirometry. Q 10 values of 1.15-2.24 were found for four of the species between 8.5-15° C, while in S. teres a Q 10 of 12.98 occurred between these temperatures. Between 15-20° C T. pyriformis and P. aurelia had Q 10 values of 3.73 and 1.56, respectively. Linear double log regressions of oxygen consumption vs. dry weight were derived at each temperature and regression coefficients (b) of 0.2723 (8.5° C), 0.4364 (15° C) and 0.4171 (20° C) were obtained. The results are explained and discussed in relation to previous work on the energetics of ciliated protozoa.