Carcinus maenas - NRC Research Press

2 downloads 0 Views 316KB Size Report
adaptation of the blood–oxygen transfer system in the crab Carcinus maenas during ... étudié chez le crabe Carcinus maenas le système d'échanges gazeux au ...

Color profile: Disabled Composite Default screen

784

Blood oxygen requirements in resting crab (Carcinus maenas) 24 h after feeding Alexia Legeay and Jean-Charles Massabuau

Abstract: Numerous resting unfed water-breathers have a strategy of gas-exchange regulation that consists of setting the arterial partial pressure of oxygen (PaO2) at 1–3 kPa. This raises a question concerning the extent to which physiological functions are limited in this situation. To obtain insight into this problem, we studied the steady-state adaptation of the blood–oxygen transfer system in the crab Carcinus maenas during the doubling of oxygen consumption, M& O2 (i.e., during the period of specific dynamic action of food (SDA)), that occurs 24 h after feeding. We showed that this increase in M& O2 24 h after a meal is not limited by a blood partial pressure of oxygen (PO2) as low as 0.8–1.5 kPa in either normoxia or hypoxia (PO2 of the inspired water = 4 kPa). In normoxia, adaptation of the oxygen-transport system, if any, consisted of a combined set of adaptations of small amplitude (in absolute value), rather than major changes in blood oxygenation status, blood flow rate, or oxygen affinity (although blood pH decreases). In hypoxia, the SDA was mainly associated with an increase in blood flow rate and blood pH, with no changes in blood lactate, urate, calcium, and haemocyanin concentrations. The results are discussed, in an environmental context, in terms of minimal oxygen requirements in water-breathers. Résumé : Chez de nombreux animaux aquatiques à jeun et au repos il existe une stratégie respiratoire qui consiste à maintenir la pression partielle d’oxygène dans le sang artériel (PaO2) à une valeur faible et constante égale à 1–3 kPa. Cela pose la question de la limitation de fonctions physiologiques in vivo. Pour approfondir ce problème, nous avons étudié chez le crabe Carcinus maenas le système d’échanges gazeux au cours du doublement de la consommation d’oxygène (M& O2) qui accompagne l’action dynamique spécifique (SDA) 24 h après une prise de nourriture. Les expériences ont été faites en normoxie et en hypoxie. Nous montrons qu’à ce moment particulier, 24 h après une prise de nourriture, M& O2 n’est pas limité par des PO2 = 0.8–1.5 kPa. En normoxie, l’adaptation des échanges gazeux est le résultat d’un ensemble d’ajustements de faible amplitude, à la limite des seuils de détection, sans changement particulièrement important de l’état d’oxygénation du sang, du débit cardiaque ou de l’affinité du sang pour l’oxygène. En hypoxie, la SDA est associée à une augmentation du débit cardiaque et du pH sanguin sans changement des concentrations de lactate, urate, calcium et hémocyanine. Les résultats sont examinés, dans un contexte écologique, en terme de besoins minimaux en oxygène chez les animaux aquatiques. 794

Introduction In water-breathing animals, including various teleosts, crustaceans, and molluscs, at rest and unfed, the existence of a strategy of tissue oxygenation that maintains the partial pressure of oxygen (PO2) in the arterial blood at low values and within a narrow range (1–3 kPa) independently of the PO2 of the inspired water (inspired PO2), which ranges from 3 to 40 kPa, has been reported under both laboratory (Massabuau and Burtin 1984; Forgue et al. 1989, 1992a; Massabuau et al. 1991) and field (Massabuau and Forgue 1996) conditions. Since numerous O2-dependent metabolic processes, including mitochondrial respiration and various enzyme reactions, exhibit O2-affinity constants that are close to or higher than the corresponding O2 concentrations (see Received June 12, 1998. Accepted January 27, 1999. A. Legeay and J.-C. Massabuau.1 Laboratoire d’Ecophysiologie et d’Ecotoxicologie des Systèmes Aquatiques, Unité Mixte de Recherche 5805, Université Bordeaux I, and Centre National de la Recherche Scientifique, Place du Docteur B. Peyneau, 33120 Arcachon, France. 1

Author to whom all correspondence should be addressed (e-mail: [email protected]).

Can. J. Zool. 77: 784–794 (1999)

J:\cjz\cjz77\cjz-05\Z99-021.vp Monday, October 04, 1999 12:26:53 PM

Jones et al. 1985; Connett al. Massabuau 1990; Vanderkooi et al. Legeayetand 1991), an important question arises: does blood PO2 limit cellular activities in water-breathers, even under normoxic environmental conditions? In accord with Jones et al. (1985), we use the term neahypoxia, or near hypoxia, for this particular oxygenation status in the internal milieu, where blood PO2 can induce reversible changes in cellular metabolism and function. Using the stomatogastric nervous system of the lobster Homarus gammarus in vitro and in vivo, we have already studied how neural networks operate under such near-hypoxic conditions. We showed that PO2 changes in the physiological range (i) can indeed limit the rapidly cycling activity of neural circuits and (ii) influence and modify the pattern of neural network activities, as well as neural network interactions, in a manner equivalent to that of a neuromodulator. Actually, 2 h after food intake (i.e., during the first stage of the digestive process in crustaceans; Hopkin and Nott 1980), a transient increase in arterial PO2 (PaO2) from 1–2 to 2–4 kPa actively participates in modulating the filtering function of the pylorus and coordinating it with the masticatory function of the gastric mill (Massabuau and Meyrand 1996; Clemens et al. 1998). The main objective of the present work was to study whether a PaO2 of 1–2 kPa also limits the increase in O2 consumption rate (M& O2) that occurs during the second stage © 1999 NRC Canada

Color profile: Disabled Composite Default screen

Legeay and Massabuau

of the digestive cycle (12–48 h after feeding), when the food is no longer being ground up in the gastric mill. In an attempt to resolve this question, we studied the O2-transport strategy that occurs 24 h after feeding in the crab Carcinus maenas. The experiments were performed in normoxia, where the blood PO2 was free to increase, and in hypoxia, where a rise in blood PO2 was prevented. Carcinus maenas was chosen for four main reasons. First, it is representative of water-breathers that live with low blood PO2 (Forgue et al. 1992a, 1992b; Massabuau and Forgue 1996). Second, the doubling of its O2 consumption after feeding is quite specific, as the crab remains inactive. Moreover, its digestive cycle has already been extensively analyzed (Hopkin and Nott 1980). Third, it inhabits ecological niches where shortterm and (or) long-term hypoxic events are regularly observed (Truchot and Duhamel-Jouve 1980). Finally, owing to its widespread invasion of new habitats, it has come to be considered a nuisance species worldwide. We show here that if any adaptation of the O2-transport system occurs during the doubling of O2 consumption 24 h after food intake in normoxia, it is of rather small amplitude at best. In any case, a PaO2 as low as 1–2 kPa represents a head of pressure high enough to fuel the oxidative reactions at the O2-demanding site(s). This allows the postprandial rise in M& O2 at +24 h to be expressed even in hypoxic water, where the inspired PO2 is 4 kPa. The postprandial increase in O2 consumption corresponds to an increase in metabolic rate that has been called the specific dynamic action of food (SDA) (Brody 1945; Beamish 1974; Jobling 1981). For reference, 1 kPa = 7.5 torr or mmHg, and in water, a PO2 of 1 kPa corresponds to an O2 fraction of ≈1% and an O2 concentration of ≈0.5 mg·L–1 at 15°C.

Materials and methods Animals and ambient conditions Experiments were performed from December to June on 240 male C. maenas in the intermolt stage that weighed 61 ± 3 g (mean ± 1 SE; range 39–90 g). They were collected locally in the Bay of Arcachon, and stocked year-round in large tanks at temperatures ranging from 8 to 20°C depending on the season. They were supplied with aerated seawater (salinity 30–32‰, pH 8.3–8.4) and fed twice weekly with fish. Before the experiments, they were acclimated in the laboratory for 1 week. They were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. At least 3 days before the first experiment, animals were transferred to the experiment room and adapted to the following conditions: temperature 15.0 ± 0.5°C; PO2 20–21 kPa; partial pressure of carbon dioxide (PCO2) 0.1 kPa; pH ≈7.76 ± 0.05 depending on the water titration alkalinity. An approximate doubling of the metabolic rate following ingestion of food was induced in the present experiment by providing mussel flesh (one mussel, fresh mass ≈2 g) per meal to each crab). Two types of analyses were performed separately under pre- and post-prandial conditions, first in normoxia and then in hypoxia, at a water PO2 of 4 kPa (i.e., 30 torr or an O2 concentration of 2 mg·L –1 in full seawater): measurement of changes in O 2 consumption rates and changes in blood-gas composition. Hypoxic water conditions were obtained by bubbling a nitrogen–air mixture through seawater via mass flow controllers (Model FC-260, Tylan General) driven by a laboratory-constructed programmable control unit.

785

Measuring oxygen consumption rates These experiments were performed in December and January on 7 crabs that weighed 49 ± 8 g (range 39–60 g). M& O2 (µmol·min–1·kg–1) was measured in an open-flow respirometer (volume 160 mL). The technique used was basically similar to that described by Massabuau et al. (1984); its main features are as follows: (i) the respirometer is equipped with a pH–CO2 stat; (ii) a rotor ensures homogeneous composition in the system (so that the water exiting the chamber, the composition of which is controlled, is closely representative of the water inspired by the crab); and (iii) a laboratorymade automatic device continuously monitors PO2 of the exit water and adjusts water flow through the respirometer to maintain the PO2 inspired by the animal at either 21 or 4 kPa. Consequently, the inspired PO2 was maintained within a narrow range (±0.5 kPa) independently of the changes in M& O2 induced by the experimental conditions. For water-chemistry adaptation, each crab was first placed in an open tank (35 L, renewal rate 0.5 L·min–1) filled with water that had been equilibrated to a PO2 of 21 kPa and a PCO2 of 0.1 kPa. From 09:00 to 09:30, it was transferred, within 2–3 min, to the respirometer (at time t0), where the PO2 was either 21 or 4 kPa. Reference preprandial M& O2 measurements were systematically followed in the afternoon (t0 + 6 h) and the next morning, from 09:00 to 10:00 (t0 + 23–24 h). Animals were fed at to + 24 h 30 min (from 10:00 until 10:30). For feeding, and to avoid contamination in the respirometer, they were temporarily transferred to a feeding chamber for 30 min (volume 400 mL; no change in water physicochemistry). They were then transferred back to the respirometer, within about 2–3 min, and measurements were performed 6 h later (from 16:30 to 17:30) and the following morning from 9:00 to 10:00, i.e., 24 h after the meal. It should be noted that, consequently, steady-state measurements were performed on resting animals that had not been handled for at least 6 h and that PO2 was measured automatically with electrodes manipulated by remote control to ensure nonstressful conditions.

Blood-gas status: experimental protocol Blood-gas analyses were performed from April to January on 226 animals weighing 61 ± 2 g (range 40–86 g). They were kept in 35-L tanks in which the water was renewed at the rate of about 0.5 L·min–1. The acid–base balance in the water was adjusted by a pH–CO2 stat (pH ≈ 7.76 ± 0.05; PCO2 = 0.1 kPa). In hypoxic water, care was taken to prevent crabs reaching the water surface and breathing air. Blood-gas analyses in pre- and post-prandial conditions were performed in three experimental series. In series 1, bloodoxygenation status was studied in pre- and post-prandial conditions in 40 crabs randomly divided into four groups. The animals were acclimated to either normoxic or hypoxic conditions for 2 or 3 days. Two groups were used as controls and two groups were fed and sampled 24 h later. In series 2, the blood acid–base balance was studied in a total of 130 crabs (10 groups of 12–14 animals each), either in normoxia (control and recovery groups) or during a 15-day period in hypoxia. In hypoxia, food was given at t0 + 1, + 7, and + 15 days (t0 is the beginning of exposure to hypoxia) and sampling was performed 24 h later, i.e., at t0 + 2, + 8, and + 16 days. During the recovery period, crabs were sampled 8 days after they were returned to normoxia. Only one group of animals was studied at each sampling time. Finally, in series 3, the role of urate concentration versus blood pH and blood PO2 was analyzed specifically (n = 56 animals). As in series 1, crabs were randomly divided into four groups. They were acclimated to either normoxic or hypoxic conditions for 2–3 days. Two groups were used as controls and two groups were fed and sampled 24 h later.

Blood-sampling method For sampling arterial blood a 1- to 2-mm hole was drilled in the shell at the level of the heart at least 5 days before measurements began and a thin layer of cuticle was left in place. A piece of rub© 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:01 PM

Color profile: Disabled Composite Default screen

786

Can. J. Zool. Vol. 77, 1999

ber was glued over the hole. Blood samples were collected by removing crabs from the water and using a capillary glass tube equipped with a needle to puncture (i) the rubber membrane to obtain arterial blood and (ii) the base of a walking leg to obtain venous blood. The arterial sample (200 µL) was obtained within the first 30 s of emersion and the venous sample (150 µL) within the first minute. With this technique, the arterial blood enters the tube spontaneously and heartbeats are clearly visible. This sampling technique was critically assessed in Forgue et al. (1992b) and Massabuau and Forgue (1996). After sampling, the blood was stored on ice and analyzed within 1–10 min.

The largest crabs (60–90 g) used for blood-gas measurements were not used to determine M& O2. Consequently, results of V&b calculations were compared when using CaO2 and CvO2 values determined from (i) crabs weighing 40–60 g and (ii) the largest specimens, which weighed 60–90 g. As no differences were observed, data were pooled. Finally, blood O2 capacitance, βa,vO2 (µmol·L–1·kPa–1), was calculated for each individual from the simultaneous measurements of PaO2, PvO2, CaO2, and CvO2 as ∆Ca,vO2/∆Pa,vO2. When included in eq. 3, this gives

Analyses of blood gases, pH, Cu and urate concentrations, and blood flow

where V&b · βa,vO2 (µmol·kPa–1·kg–1·min–1) is the perfusive conductance (Dejours 1981). When the sampling required for the above analyses was performed, an additional ≈500 µL of venous blood was taken, frozen, and used to determine the blood lactate concentration, [lact]b (Boehringer, kit No. 139084; see Gäde 1984), and [Ca]b (Boehringer, kit No. 1273574) on subsequent days.

Venous blood pH was measured in 75-µL samples using a capillary G299A electrode thermostatically controlled at 15°C; total CO2 concentration (µmol·L–1) in the venous blood was measured in 25-µL samples using a modified Cameron chamber (Cameron 1971). Bicarbonate plus carbonate concentrations (mmol·L–1) and venous PCO2 (PvCO2) (kPa) were calculated using a CO2 solubility of 374 µmol·L–1·kPa–1, a pK′1 of 6.027, and a pK′2 of 9.29 (Truchot 1976). Throughout the text, HCO3–v corresponds to HCO3–v + CO32 –v. PaO2 and venous PO2 (PvO2) (kPa) were determined within 3 min on 100-L samples using a E5046 radiometer polarographic electrode thermostatically controlled at 15°C and equipped with a thin radiometer head cover to reduce dead space. The electrode was calibrated with seawater that had been equilibrated with O2free N2 and low PO2 standards (O2 fraction, FO2 = 2.35 or 4.44%) obtained via mass flow controllers (Model FC-260, Tylan General). The gas-phase composition of these standards was regularly controlled using a paramagnetic O2 analyzer (Servomex 1100A) calibrated with a high-grade gas mixture (FO2 = 3.99 ± 0.04%). As shown in Massabuau and Forgue (1996, Fig. 2), the present calibration procedure, which improves analytical quality in the low range, does not preclude the reading of high blood PO2 values. The blood O2 concentration, CO2 (µmol·L–1), was determined using a modified Tucker chamber (Tucker 1967) thermostatically controlled at 37°C and calibrated with various volumes of normoxic distilled water (10–50 µL). Potassium cyanide (6 g·L–1) was used for releasing bound O2 from haemocyanin (Bridges 1983). The blood haemocyanin concentration, [Hc]b (g·L–1), was calculated after the blood copper concentration, [Cu]b, was measured (Boehringer, kit No. 124834), assuming that Hc contains 0.173% copper (Truchot 1978). The maximum O2-carrying capacity, CHcO2max (µmol·L–1), was calculated from each individual [Cu]b using the experimental equation given in Truchot (1978) for C. maenas:

[1]

CHcO2max = 0.4505 [Cub] – 0.0384

The dissolved O2 concentration, CO2 diss (µmol·L–1), in the arterial and venous blood was calculated using an O2-solubility coefficient of 10.5 µmol·L–1·kPa–1 (Truchot 1978). The corresponding saturation, SO2 (%), was estimated for each individual:

[2]

SO2 = ((CO2 total – CO2

diss )

CHcO2max–1) · 100

The urate concentration, [urate]b (µmol·L–1), was analyzed in blood-sample replicates, following the protocol of Lallier and Truchot (1989; Sigma, diagnostic kit No. 685). The circulatory blood flow rate, V&b (mL·min–1·kg–1), for each crab was calculated using the Fick principle (Dejours 1981), paired differences in arteriovenous O2 concentration (µmol·L–1), and mean M& O2 (µmol·min–1·kg–1) measured, for technical reasons, in a different batch of crabs:

[3]

M& O2 = V& b (CaO2 – CvO2)

[4]

M& O2 = V& b · βa,vO2 · (PaO2 – PvO2)

Statistics All data are presented as individual values in histograms of frequency distribution and (or) as means ± 1 standard error (SE). Paired t tests were used to analyze changes in M& O2, blood O2 status, and acid–base balance before and after feeding. Otherwise, differences between normoxic and hypoxic conditions were evaluated using the Mann–Whitney U test or Student’s t test. P < 0.05 was taken as the fiducial limit of significance. Acid–base balance states were considered to be distinct when two parameters out of three (pH, HCO3, and PvCO2) were significantly different.

Results Blood oxygenation status during the postprandial & O in normoxic C. maenas at rest increase in M 2 When resting crabs were starved for 7 days, adapted in the respirometer for 24 h, and fed mussel flesh under normoxic conditions, their M& O2 increased as shown in Fig. 1A (see also Table 1). Six hours after feeding (the vertical broken line in Fig. 1A indicates the feeding time), a plateau value was reached and maintained for 24 h. Twentyfour hours after feeding, M& O2 had increased 1.8 ± 0.2 times above resting values. The period before feeding, during which crabs were simply transferred from the feeding chamber to the respirometer and were not fed (see Materials and methods), corresponds to a control. This transfer did not induce any significant change in M& O2. The corresponding blood oxygenation status is presented in Figs. 1B, 1C, and 1D and the associated changes in the calculated blood flow rate are presented in Fig. 1E. The period before feeding was characterized by the main occurrence of low blood PO2 values (Fig. 1B1 and Table 1). Most PaO2 values were in the range 0.50–1.50 kPa, with PvO2 values distributed from 0.25 to 1.25 kPa. The distribution of the arterial O2 concentrations was also quite homogeneous below 200 µmol·L–1, with only a few values as high as ≈600 µmol·L–1. Twenty-four hours after feeding (Fig. 1B2 and Table 1), three striking observations were made: there was no significant rise in PaO2 and (or) PvO2 (except in 2 individuals out of 39), a blood acidosis occurred, and arterial and venous blood O2 concentrations increased toward the higher range. It should be noted that although the analyses were performed in a large number of crabs (n = 39 in three © 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:04 PM

Color profile: Disabled Composite Default screen

Legeay and Massabuau

787

Table 1. Respiratory variables in crabs (Carcinus maenas) exposed to various oxygenation levels in preprandial and postprandial (24 h after being fed a mussel) conditions.

M& O2 (µmol·min–1·kg–1) PaO2, mean (kPa) PaO2, mode (kPa) PvO2 (kPa) ∆Pa,vO2 (kPa) CaO2 (µmol·L–1) CvO2 (µmol·L–1) ∆Ca,vO2 (µmol·L–1) [Hc]b (g·L–1) SaO2 (%) SvO2 (%) βa,vO2 (µmol·L–1·kPa–1) EbO2 V&b (mL·min–1·kg–1) V&b·βa,vO2 (µmol·kPa–1·kg–1·min–1) pHv CvCO2 (mmol·L–1) [HCO3–]v (mmol·L–1) PvCO2 (kPa) [Lact]b (mmol·L–1) n[lact]b ≤ [lact]b control [Ca]b (mmol·L–1) [urate]b (µmol·L–1)

Inspired PO2 = 20.2±0.3 kPa

Inspired PO2 = 4.0±0.4 kPa

Preprandial

Postprandial

Preprandial

Postprandial

14.81±0.66 (7) 1.05±0.09 (39) 0.75–1.00 0.54±0.04 (39) 0.51±0.07 (39) 141±18 (37) 44±7 (36) 97±13 (36) 31±3 (12) 39±5 (36) 12±2 (36) 254±33 (33) 0.68±0.03 (36) 232±24 (35) 41±5 (35) 7.79±0.01 (23) 9.21±0.44 (23) 9.05±0.43 (23) 0.41±0.02 (23) 0.07±0.04 (23) — 11.88±0.48 (10) 85±5 (24)

26.53±2.71* (7) 1.68±0.33 (39) 0.75–1.00 0.66±0.09 (39) 1.02±0.25 (39) 168±19 (38) 66±8* (31) 126±17 (31) 28±3 (12) 48±6 (37) 21±2* (27) 237±35 (31) 0.63±0.04 (31) 284±34 (28) 61±9 (28) 7.65±0.04* (22) 8.80±0.41 (22) 8.57±0.40 (22) 0.55±0.07* (22) 0.42±0.15 (21) 16 10.60±0.33 (10) 57±3* (24)

13.40±0.39 (5) 0.73±0.03* (33) 0.50–0.75 0.36±0.02* (33) 0.37±0.02 (33) 148±13 (33) 19±5* (29) 135±14* (29) 29±3 (12) 41±4 (33) 8±3 (16) 409±52* (29) 0.87±0.04* (29) 127±16* (28) 41±3 (27) 7.96±0.02* (17) 5.64±0.40* (17) 5.58±0.39* (17) 0.18±0.02* (17) 0.23±0.08 (30) 28 11.10±0.20 (10) 158±9* (24)

24.32±1.14* (5) 0.78±0.03* (33) 0.75–1.00 0.39±0.03* (33) 0.39±0.02 (33) 161±14 (33) 26±4‡ (32) 138±14* (32) 29±3 (12) 46±4 (33) 8±2 (26) 405±45* (32) 0.82±0.03‡ (32) 219±20† (31) 71±5‡ (31) 8.01±0.02* (14) 6.78±0.45* (14) 6.83±0.47* (14) 0.19±0.01* (14) 2.52±1.26 (28) 23 10.56±0.22 (10) 199±21* (24)

Note: Values are given as the mean ± SE, with the number of animals tested in parentheses. A constant acid–base balance and a temperature of 15°C were maintained in the water. Mean body mass of crabs was 67 ± 3 g (range 39–90 g). Inspired PO2 is the O2 partial pressure in the inspired water; M& O2 is the O2 consumption rate per kilogram of body mass; PaO2 is PO2 in the arterial blood expressed as the mean or mode value; PvO2 is PO2 in the venous blood; ∆Pa,vO2 is the PO2 difference between arterial and venous blood; CaO2 is the O2 concentration in the arterial blood; CvO2 is the O2 concentration in the venous blood; ∆Ca,vO2 is the CO2 difference between arterial and venous blood; [Hc]b is the haemocyanin blood concentration; SaO2 is arterial O2 saturation; SvO2 is venous O2 saturation; βa,vO2 is blood O2 capacitance; EbO2 is the extraction coefficient of blood O2; V& b is the circulatory blood flow rate per unit of body mass; V& b·βa,vO2 is the perfusive conductance per unit of body mass; pHv is the pH of the venous blood; CvCO2 is the CO2 concentration in the venous blood; [HCO3–]v is the HCO3 concentration in the venous blood; PvCO2 is the CO2 partial pressure in the venous blood; [lact]b is the blood lactate concentration; n[lact]b ≤ [lact]b control is the number of animals in which the blood lactate concentration did not differ from the normoxic control; [Ca]b is the calcium concentration in the blood; and [urate]b is the urate concentration in the blood. *Significantly different from preprandial normoxia. † Significantly different from preprandial hypoxia. ‡ Significantly different from preprandial normoxia and preprandial hypoxia.

experiments), most events did not reach statistical significance, except the blood acidosis, the increase in venous O2 content and O2 saturation, and the decrease in specific blood flow rate (V& b · M& O2–1 (mL·µmol–1)), i.e., the blood flow rate required to transport a unit quantity of O2 from the gills to the cells. This suggests that 24 h after feeding, the doubling of M& O2 directly attributable to the SDA either is performed without any adjustment of the O2-transport system or is the result of a combined set of adaptations of small amplitude (in absolute value) at the limit of experimental detection. However, in one of the three experimental groups (included in the above mean values), the mean PaO2 in controls was lower than in the above pooled data (0.8 ± 0.1 instead of 1.1 ± 0.1 kPa). Interestingly, it was only in this group that significant increases in PaO2 (up to 1.3 ± 0.3 kPa), O2 content (from 117 ± 27 to 204 ± 42 µmol·L–1), and O2 saturation (from 30 ± 6 to 57 ± 9%) occurred. This further illustrates the exceptionally small range within which efficient adaptation can occur in normoxic crabs during the SDA. Therefore,

we addressed the issue of whether the postprandial rise in M& O2 could also be expressed in hypoxia. & O at an inspired PO of 4 kPa Postprandial rise in M 2 2 & M O2 and blood oxygenation status at 4 kPa before feeding are shown in Fig. 2A (n = 33). Importantly, preprandial M& O2 in hypoxia was not significantly different from M& O2 during preprandial normoxia. The corresponding blood O2 status is presented in Figs. 2B, 2C, and 2D and the associated changes in the calculated blood flow rate in Fig. 2E. The striking feature here is a significant decrease in PaO2 (from ≈1 to ≈0.7 kPa) that was not accompanied by a decrease in arterial O2 content, as ventilation-induced alkalinization increased the blood O2 capacitance (∆Ca,vO2/∆Pa,vO2). Finally, it should be noted that as the venous O2 content decreased compared with the normoxic unfed status also, the difference between the venous and arterial O2 contents increased significantly, which strongly suggests that in these resting and unfed crabs the blood flow rate had decreased by 25%. © 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:08 PM

Color profile: Disabled Composite Default screen

788

Can. J. Zool. Vol. 77, 1999

Fig. 1. Characterization of oxygen consumption rates (M& O2) and blood O2 status in preprandial (after a 7-day period without food) and postprandial (24 h after a meal) crabs (Carcinus maenas) in normoxia (inspired PO2 = 21 kPa). (A) Oxygen consumption. Crabs were placed in the respirometer 24 h prior to feeding. The vertical broken line indicates the point at which they were fed. The postprandial M& O2 was 1.8 times the preprandial rate. The rate did not vary statistically between 6 and 24 h (n = 5–7 crabs; values are given as the mean ± 1 SE; paired measurements); *, significantly different from preprandial normoxia. (B1 and B2) Blood O2 status. Frequency distributions of PO2 and CO2 in the arterial (PaO2, CaO2) and venous (PvO2, CvO2) blood in preprandial (B1) and postprandial (B2) normoxia. Before and after feeding, the most frequently measured PaO2 was in the same low range, 0.50–1.25 kPa, independently of M& O2. PvO2 values were distributed from 0.25 to1 kPa. (C) Relationships between mean blood CO2 and PO2 values in normoxia before and after feeding. (D) Arteriovenous O2 content difference (∆Ca,vO2) before and after feeding. (E) Circulatory blood flow rate (V&b) before and after feeding. n = 29–39. For mean values and statistical significance see Table 1.

The change in M& O2 occurring 24 h after feeding at 4 kPa is presented at the right-hand side of Fig. 2A. It is clear that M& O2 was not O2-limited, since it increased significantly by 1.8 ± 0.1 times, a factor comparable to the normoxic control. The corresponding changes in blood oxygenation are shown in Figs. 2B and 2C. The major features are (i) remarkable

constancy of blood O2 status, with the exception of mean venous O2 content, which increased significantly, and (ii) significant blood pH alkalinization superimposed on that resulting from transfer from normoxia to hypoxia (Table 1). As this was not accompanied by any change in blood O2 capacitance, these data suggest that all adaptation depended on © 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Wednesday, October 06, 1999 12:17:48 PM

Color profile: Disabled Composite Default screen

Legeay and Massabuau

789

Fig. 2. Characterization of oxygen consumption rates (M& O2) and blood O2 status in preprandial (after a 7-day period without food) and postprandial (24 h after a meal) crabs in hypoxia (inspired PO2 = 4 kPa). (A) Oxygen consumption rates. Crabs were placed in the respirometer 24 h prior to feeding. The vertical broken line indicates the point at which they were fed. As in normoxia, postprandial M& O2 was 1.8 times the preprandial rate. The rates did not vary statistically between 6 and 24 h (n = 5–7 crabs; values are given as the mean ± 1 SE; paired measurements). (B1 and B2) Blood O2 status. Frequency distributions of PO2 and CO2 in the arterial (PaO2, CaO2) and venous (PvO2, CvO2) blood in preprandial (B1) and postprandial (B2) hypoxia. Data for postprandial crabs were determined 24 h after a meal. Before and after feeding, the most frequently measured PaO2 was in the same low range, 0.50–0.75 kPa, independently of M& O2. PvO2 values were mainly in the range 0.25–0.75 kPa. (C) Relationships between mean blood CO2 and PO2 values in hypoxia before and after feeding. Compared with normoxia, there was a global shift in PO2 to the left. (D) The arteriovenous O2 content difference (∆Ca,vO2) before and after feeding. (E) Circulatory blood flow rates (V&b) before and after feeding. In preprandial hypoxia, Vb was significantly lower than in preprandial normoxia. It increases in postprandial hypoxia. n = 29–39. For mean values and statistical significance see Table 1; *, significantly different from preprandial normoxia; **, significantly different from preprandial hypoxia.

© 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:35 PM

Color profile: Disabled Composite Default screen

790

Can. J. Zool. Vol. 77, 1999

Fig. 3. Relationships between blood O2 capacitance (βa,vO2) and various blood cofactors: haemocyanin concentration ([Hc]b) (A), calcium concentration ([Ca]b) (B), lactate concentration ([lact]b) (C), pH (D), and urate concentration ([urate]b) (E). There was a correlation only between βa,vO2, pH, and [urate]b. All values are from Table 1.

an increase in the calculated blood flow rate. It is worth noting that, following this increase, the blood flow rate was identical with that of the control in preprandial normoxia. The above results, then, show that in normoxia and hypoxia, the postprandial rise in M& O2 can be expressed with PaO2 values as low as ≈0.7–1 kPa, and they stress the role that can be played by the blood flow rate and the O2-carrying capacity of the blood. Therefore, we focused our attention next on changes in blood O2 affinity and concentrations of blood cofactors. Blood O2 capacitance (βa,vO2) changes and blood cofactors Figure 3 (and Table 1) summarizes the changes in βa,vO2 that occur in the various situations analyzed above as a function of various blood cofactors (Mangum 1983; Truchot 1992). It shows, first, that a change in βa,vO2 was only observed following the transfer from normoxia to hypoxia and not before and after feeding and, second, that there was no relationship between blood O2 capacitance and [Hc]b, [Ca]b, and [lact]b, only with blood pH and [urate]b. Figures 4A and 4B give more insight into the question of [lact]b, especially during hypoxia (Fig. 4B), by showing the distribution of individual values after feeding. It is worth noting that [lact]b increased to very high values in a few animals. With regard to [urate]b, Lallier et al. (1987) showed that it increased when the inspired PO2 decreased. Figures 4C and 4D confirm this relationship, but, interestingly, show that [urate]b is correlated with blood pH (Fig. 4C) but is independent of PaO2 status (Fig. 4D). This suggests that changes in [urate]b could be correlated with the inspired PO2 only via ventilationinduced alkalinization. As reported above, the transfer from normoxia to hypoxia in these crabs induced classic blood alkalosis, and the SDA was associated with a blood acidosis in normoxia and a blood alkalosis in hypoxia. Surprisingly, these postprandial pH changes were not associated with any visible effect on blood O2 capacitance in vivo. To obtain more insight into this question and to test further the reliability of the pH adjustments, we then studied blood pH regulation during a

series of meals (in a single experiment) before, during, and after a 15-day exposure period in hypoxia. Figure 5 shows that during the control and recovery situations in normoxia, feeding was indeed systematically accompanied by a metabolic acidosis (including a ventilatory component in the control experiment), and that in hypoxia at 4 kPa, it was systematically accompanied by a metabolic alkalosis (statistically significant in two experiments out of three). Moreover, blood pH was regulated at various but reproducible apparent set-points that depended both on the metabolic level and the inspired PO2.

Discussion In this study, we present evidence that in a water-breather, the crab C. maenas, the SDA can induce a near doubling of O2 consumption without being limited by PaO2 values as low as 0.8–1 kPa. This can be performed with a PO2 of 4 kPa in the inspired water. To our knowledge, these results, obtained in the laboratory using crabs isolated from nonspecific external stimulation, represent the first demonstration of the minimal O2 requirements, in terms of arterial O2 head pressure, for digestion 24 h after feeding in a crustacean. Moreover, our data provide insight into the framework of overall blood respiratory adjustments (O2, V& b, and blood acid–base balance) required to achieve this specific increase in oxidative metabolism. Many of the adjustments, when present, occurred at extremely low values and within a narrow range, at the limits of experimental detection. Comparison with previous data To our knowledge, adaptation of the O2-transfer system to specifically meet the postprandial O2 demand in waterbreathers has never been studied per se. The metabolic rate in our resting crabs was comparable to previous data in the literature (Wallace 1973; Taylor and Butler 1978; Taylor and Wheatly 1981; Forgue et al. 1992b) despite the low blood PO2 levels measured and the fact that higher metabolic rates have also been reported (see the review in McMahon and Wilkens 1983). The near doubling of M& O2 during SDA at © 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:40 PM

Color profile: Disabled Composite Default screen

Legeay and Massabuau

791

Fig. 4. Blood lactate concentration in pre- and post-prandial conditions with and without O2 limitation due to the environment. (A and B) Frequency distributions of blood lactate concentration ([lact]b) at a PO2 of 21 kPa (normoxia) and 4 kPa (hypoxia) in the inspired water. With only a few exceptions, values remained in the same low range independently of water oxygenation. n = 12. (C and D) Relationships between blood urate concentration ([urate]b) and mean arterial pH or most frequently measured PaO2 (mode from Fig. 2). [Urate]b was dependent on blood pH, but varied independent of PaO2. Data are from Table 1; values are given as the mean ± 1 SE; *, significantly different from the normoxic preprandial control.

+24 h agrees with the classical value for most water-breathers studied (Jobling 1981; Houlihan et al. 1990). The low blood PO2 values we report were in the same low range as those we found previously for C. maenas (Massabuau and Forgue 1996), but no others have been reported in the literature (see reviews by Mangum 1980; McMahon and Wilkens 1983; Shelton et al. 1986; Truchot 1992). Values for other species are given by Angersbach and Decker (1978), McMahon (1985), and Ellis and Morris (1995). McMahon (1985) and Truchot (1992) proposed that the systematic occurrence of a high blood PO2 could be related to stressful conditions. This is indeed what was reported for the first days after transfer from field to laboratory conditions (Massabuau and Forgue 1996). From this viewpoint, it is very likely that the “sensoryreduced conditions” under which we maintained the crabs in the laboratory during the present study (resting, settled animals were given with a single mussel per feeding) did not call for a higher blood PO2. Also, a PaO2 of 0.7 kPa, without the anaerobic metabolism being switched on, is consistent with previous PaO2 values obtained at the anaerobic threshold in the crabs C. maenas and Eriocheir sinensis (Forgue et al. 1992b), but is much lower than the critical pressure of 8– 10 kPa reported by Taylor (1976). The latter difference remains unexplained. With regard to cardiac output, the values calculated here are at the high end of values given in the literature, but these are generally cited for active animals (see

reviews in Mangum 1983; McMahon and Wilkens 1983; De Wachter and McMahon 1996). De Wachter and McMahon (1996) proposed that this could be due to the use of the Fick principle to calculate blood flow rates. We suggest that as all data were obtained with the same technical approach, they are valid for comparative purposes. Importantly, however, it must be stressed that when blood PO2 in crustaceans is low, O2 saturation is minimal (see Table 1), as are O2 content and the difference in arteriovenous O2 content. Therefore, following eq. 3, the blood flow rate must be proportionally higher. Thus, notwithstanding the basic technical difficulty of measuring flow rates, it could be that the low-PO2 strategy calls for higher blood flow rates. Origin of the postprandial increase in O2 consumption 24 h after feeding Carcinus maenas was chosen partly because it is inactive after a meal. Consequently, no O2 consumption related to exercising was superimposed on the postprandial increase in O2 consumption. The causes of this increase were discussed by Jobling (1981) for fishes and studied by Houlihan et al. (1990) in C. maenas. To summarize, Houlihan et al. (1990) showed that in C. maenas, (i) the minimum cost of protein synthesis accounted for 20–37% of postprandial M& O2 and (ii) the protein-synthesis rate in the hepatopancreas, gill, heart, proventriculus, and leg extensor muscle closely paral© 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:47 PM

Color profile: Disabled Composite Default screen

792

Can. J. Zool. Vol. 77, 1999

Fig. 5. [HCO3–] plotted against pH, showing the time course of changes in blood acid–base balance in crabs before and after (+24 h) a series of consecutive meals in normoxia during a 15-day exposure period in hypoxia (inspired PO2 = 4 kPa) and after 8 days of recovery in normoxia. Feeding in normoxia systematically induced blood pH acidification, while it systematically induced alkalinization in hypoxia. Note that the pH was set at different but reproducible values depending on the four different physiological states. Pre- and post-prandial values are paired measurements. Values are given as the mean ± 1 SE; n = 12–14 crabs per data point; *, significantly different from preprandial normoxia; **, significantly different from preprandial hypoxia.

leled the postprandial increase in O2 consumption. Consequently, we suggest that the increase in O2 consumption we measured 24 h after feeding was mainly and specifically related to the intracellular digestion process. However, the comparison between Fig. 1 in the present work and Houlihan’s data (1990) shows that despite the use of similar animals and food ratio (2.5% of live crab mass), the pattern of postprandial increase in metabolism differed. Houlihan et al. (1990) reported a peak in O2 consumption 2.8 ± 0.56 h after a meal and elevation above the resting rate that ended after 10–28 h. We observed a plateau between 6 and 24 h. The reasons for this discrepancy remain unclear, although a putative explanation could be linked to a noticeable difference in experimental design between the two studies (D.F. Houlihan, personal communication) and the O2-uptake levels in control (unfed) crabs: 23.2 ± 1.2 µmol·kg–1·min–1 in Houlihan et al. (1990) versus 14.8 ± 0.7 µmol·kg–1·min–1 in this study (Table 1). Diffusive conductance of O2 at the cellular level In the present study, adaptation of perfusive conductance, which allows postprandial transport of O2 from the gills to the cells, was investigated. It must now be explained how O2 influx by diffusion into O2-demanding cells could double while PaO2 remained constant at ≈0.75 kPa (Fig. 2). At the cellular level, once the blood O2 concentration is sufficient,

i.e., once there is a large enough O2 reservoir in the vicinity of the cell, the rate of O2 transfer from the arterial capillaries to the cells depends on the diffusive conductance and on the difference between the mean O2 partial pressure in the systemic blood capillaries and the tissues. The present results demonstrate the existence of a sufficiently low diffusive resistance at the O2-demanding site(s), as a low PaO2 provides a large enough head of pressure. In this respect, the crustacean hepatopancreas, which is obviously greatly involved in the increase in O2 consumption 24 h after feeding, appears to be an exceptionally well vascularized organ, where the barrier to O2 diffusion is reduced to a minimum. Indeed, this organ is composed of thousands of blind-ending tubules that consist of a single layer of epithelial cells bordered by a discontinuous network of contractile cells (for the lobster see Fig. 23c in Icely and Nott 1992; for C. maenas see Stanier et al. 1968). These tubules are bathed in the sinuses, where numerous terminal hepatic arterioles directly discharge arterial blood (Factor and Naar 1990). These anatomical observations, and the low blood PO2 we report, are consistent with a previous study by Lallier and Walsh (1990), who demonstrated the absence of any limiting effect of a PO2 as low as 2 kPa on the metabolism of isolated hepatopancreas cells from the blue crab (Callinectes sapidus). Finally, it is important to note that present observations reinforce the conclusion of McMahon and Burnett (1990) © 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:33:58 PM

Color profile: Disabled Composite Default screen

Legeay and Massabuau

that the open circulatory system of crustaceans is a welldesigned apparatus allowing highly efficient tissue perfusion. Blood pH changes 24 h after feeding The results of the present study show that the SDA 24 h after feeding was systematically associated with a blood acidosis in normoxia and a blood alkalosis in hypoxia (Fig. 5). However, we did not observe any associated change in blood O2 affinity, although (i) in hypoxia, the alkalosis could have favoured O2 loading at the gill level, and (ii) in normoxia, the acidosis could have favoured O2 unloading at the cellular level, in conditions where the inspired PO2 was not limiting. Two alternative but not mutually exclusive explanations are possible. First, the small amplitude of the pH change (≈0.05– 0.10 pH units) induced a change in blood O2 affinity that was below our detection limits. Second, the blood pH change participated in the modulation of some undetermined pHsensitive metabolic reaction. Importantly, however, it must be stressed again that the postprandial rise in M& O2 was expressed despite the absence of an observable change in blood O2 affinity. Consequently, this reinforces the notion that 24 h after a meal, M& O2 can double even without a large increase in blood CO2. Conclusions In summary, based on the experimental evidence from the present study and the recent work of Clemens et al. (1998) on the blood PO2 required during the first steps of the digestive process in the lobster H. gammarus, this report shows how postprandial O2 requirements are met within the framework of the low blood PO2 strategy in water breathers described above (see Introduction). We have shown that in C. maenas, 24 h after feeding, regardless of whether the water PO2 is 21 or 4 kPa, PaO2 is set at 1–1.5 kPa, which is high enough to allow the near doubling of the postprandial & MO 2 classically observed at that time. Whether the efficiency of the intracellular digestive process is optimal (in terms of protein synthesis, for example) in this condition remains to be studied; nevertheless, these results yield new insights into the oxygenation status at which cells function in situ to perform the corresponding metabolic reactions. We suggest that this could be used as a guideline for further study of feeding in these animals and, moreover, that it will shed new light on our approach to analyzing problems associated with low-oxygenated waters in the field.

Acknowledgements The authors thank Dr. J. Forgue and Prof. D. Houlihan for critical reading of the manuscript and P. Ciret for his technical assistance. Special thanks are extended to R.F. Burton for his comments. A.L. was supported by a grant from the French Ministry of Research and Education.

References Angersbach, D., and Decker, H. 1978. Oxygen transport in crayfish blood: effect of thermal acclimation, and short term fluctuations related to ventilation and cardiac performance. J. Comp. Physiol. B, 123: 105–112.

793 Beamish, F.W.H. 1974. Apparent specific dynamic action of largemouth bass, Micropterus salmoides. J. Fish. Res. Board Can. 31: 1763–1769. Bridges, C.R. 1983. PO2 and oxygen content measurement in blood samples using polarographic oxygen sensors. In Polarographic oxygen sensors. Edited by E. Gnaiger and H. Forstner. SpringerVerlag, Heidelberg. pp. 219–233. Brody, E. 1945. Bioenergetics and growth. Reinolds, New York. Cameron, J.N. 1971. Rapid method for determination of total carbon dioxide in small samples. J. Appl. Physiol. 31: 632–634. Clemens, S., Massabuau, J.-C., Legeay, A., Meyrand, P., and Simmers, J. 1998. Interaction of two interacting central pattern generators in lobster stomatogastric ganglion: influence of feeding and local partial pressure of oxygen. J. Neurosci. 18: 2788–2799. Connett, R.J., Honig, C.R., Gayeski, T.E.J., and Brooks, G.A. 1990. Defining hypoxia: a systems view of VO2, glycolysis, energetics, and intracellular PO2. J. Appl. Physiol. 68: 833–842. Dejours, P. 1981. Principles of comparative respiratory physiology. Elsevier, Amsterdam, New York, and Oxford. De Wachter, B., and McMahon, B.R. 1996. Haemolymph flow distribution, cardiac performance and ventilation during moderate walking activity in Cancer magister (Dana) (Decapoda, Crustacea). J. Exp. Biol. 199: 627–633. Ellis, B.A., and Morris, S. 1995. Effects of extreme pH on the physiology of the Australian “yabby” Cherax destructor: acute and chronic changes in haemolymph oxygen levels, oxygen consumption and metabolite levels. J. Exp. Biol. 198: 409–418. Factor, J.R., and Naar, M. 1990. The digestive system of the lobster, Homarus gammarus: II. Terminal hepatic arterioles of the digestive gland. J. Morphol. 206: 283–291. Forgue, J., Burtin, B., and Massabuau, J.-C. 1989. Maintenance of oxygen consumption in resting Silurus glanis at different levels of ambient oxygenation. J. Exp. Biol. 143: 305–319. Forgue, J., Truchot, J.-P., and Massabuau, J.-C. 1992a. Low arterial PO2 in resting crustaceans is independent of blood oxygen affinity. J. Exp. Biol. 170: 257–264. Forgue, J., Massabuau, J.-C., and Truchot, J.-P. 1992b. When are resting water breathers lacking O2? Arterial PO2 at the anaerobic threshold in crab. Respir. Physiol. 88: 247–256. Gäde, G. 1984. Effects of oxygen deprivation during anoxia and muscular work on the energy metabolism of the crayfish, Orconectes limosus. Comp. Biochem. Physiol. A, 77: 495–502. Hopkin, S.P., and Nott, J.A. 1980. Studies on the digestive cycle of the shore crab Carcinus maenas (L.) with special reference to the B cells in the hepatopancreas. J. Mar. Biol. Assoc. U.K. 60: 891–907. Houlihan. D.F., Waring, C.P., Mathers. E., and Grey, C. 1990. Protein synthesis and oxygen consumption of the shore crab Carcinus maenas after a meal. Physiol. Zool. 63: 735–756. Icely, J.D., and Nott, J.A. 1992. Digestion and absorption: digestive system and associated organs. In Microscopic anatomy of invertebrates. Vol. 10. Decapod Crustacea. Edited by F.W. Harrison. Wiley–Liss, Inc., New York. pp. 147–201. Jobling, M. 1981. The influences of feeding on the metabolic rate of fishes: a short review. J. Fish Biol. 18: 385–400. Jones, D.P., Kennedy, F., Andersson, B.S., Aw, T.Y., and Wilson, E. 1985. When is a mammalian cell hypoxic? Insights from studies of cells versus mitochondria. Mol. Physiol. 8: 473–482. Lallier, F., and Truchot, J.P. 1989. Hemolymph oxygen transport during environmental hypoxia in the shore crab, Carcinus maenas. Respir. Physiol. 77: 323–336. Lallier, F., and Walsh, P.J. 1990. Metabolism of isolated hepatopancreas cells from the blue crab (Callinectes sapidus) under © 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:34:01 PM

Color profile: Disabled Composite Default screen

794 stimulated postexercise and hypoxic conditions. Physiol. Zool. 65: 712–723. Lallier, F., Boitel, F., and Truchot, J.P. 1987. The effect of ambient oxygen and temperature on haemolymph L-lactate and urate concentrations in the shore crab Carcinus maenas. Comp. Biochem. Physiol. A, 86: 255–260. Mangum, C.P. 1980. Respiratory function of the hemocyanins. Am. Zool. 20: 19–38. Mangum, C.P. 1983. Oxygen transport in the blood. In The biology of Crustacea. Vol. 5. Edited by L.H. Mantel. Academic Press, New York and London. pp. 289–372. Massabuau, J.-C., and Burtin, B. 1984. Regulation of oxygen consumption in the crayfish Astacus leptodactylus at different levels of oxygenation: role of peripheral O2 chemoreception. J. Comp. Physiol. B, 155: 43–49. Massabuau, J.-C., and Forgue, J. 1996. A field versus laboratory study of blood oxygen status in normoxic crabs at different temperatures. Can. J. Zool. 74: 423–430. Massabuau, J.-C., and Meyrand, P. 1996. Modulation of a neural network by physiological levels of oxygen in lobster stomatogastric ganglion. J. Neurosci. 16: 3950–3959. Massabuau, J.-C., Dejours, P., and Sakakibara, Y. 1984. Ventilatory CO2 drive in the crayfish: influence of oxygen consumption and water oxygenation. J. Comp. Physiol. B, 154: 65–72. Massabuau, J.-C., Burtin, B., and Wheatly, M. 1991. How is O2 consumption maintained independent of ambient oxygen in mussel Anodonta cygnea? Respir. Physiol. 83: 103–114. McMahon, B.R. 1985. Functions and functioning of crustacean hemocyanin. In Respiratory pigments in animals. Relation structure function. Edited by J.N. Lamy, J.P. Truchot, and R. Gilles. Springer-Verlag, New York. pp. 35–58. McMahon, B.R., and Burnett, L.E. 1990. The crustacean open circulatory system: a reexamination. Physiol. Zool. 63: 35–71. McMahon, B.R., and Wilkens, J.L. 1983. Ventilation, perfusion and oxygen uptake. In The biology of Crustacea. Vol. 5. Edited by L.H. Mantel. Academic Press, New York and London. pp. 289–372. Shelton, G., Jones, D.R., and Milsom, W.K. 1986. Control of

Can. J. Zool. Vol. 77, 1999 breathing in ectothermic vertebrates. In Handbook of physiology. Sect. 3. The respiratory system. Edited by A.P. Fishman. The American Physiological Society and The Williams and Wilkins Co., Baltimore, Md. pp. 857–909. Stanier, J.E., Woodhouse, M.A., and Griffin, R.L. 1968. The fine structure of the hepatopancreas of Carcinus maenas (L.) (Decapoda Brachyura). Crustaceana (Leiden), 14: 56–66. Taylor, A.C. 1976. The respiratory responses of the crab Carcinus maenas to declining oxygen tension. J. Exp. Biol. 65: 309–322. Taylor, E.W., and Butler, P.J. 1978. Aquatic and aerial respiration in the shore crab, Carcinus maenas (L.), acclimated to 15°C. J. Comp. Physiol. B, 127: 315–323. Taylor, E.W., and Wheatly, M. 1981. Ventilation, heart rate and respiratory gas exchange in the crayfish Austropotamobius pallipes (Lereboullet) submerged in normoxic water and following 3 h exposure in air at 15°C. J. Comp. Physiol. B, 138: 67–78. Truchot, J.P. 1976. Carbon dioxide combining properties of the blood of the shore crab Carcinus maenas (L.): carbon dioxide solubility coefficient and carbonic acid dissociation constants. J. Exp. Biol. 64: 45–57. Truchot, J.P. 1978. Variations de la concentration sanguine d’hémocyanine fonctionnelle au cours du cycle d’intermue chez le crabe Carcinus maenas (L.). Arch. Zool. Exp. Gen. 119: 265–282. Truchot, J.P. 1992. Respiratory function of arthropod hemocyanins. In Advances in comparative and environmental physiology. Vol. 13. Edited by C.P. Mangum. Springer-Verlag, Heidelberg. pp. 378–410. Truchot, J.P., and Duhamel-Jouve, A. 1980. Oxygen and carbon dioxide in the marine intertidal environment: diurnal and tidal changes in rockpools. Respir. Physiol. 39: 241–254. Tucker, V.A. 1967. Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23: 410–414. Vanderkooi, J.M., Erecinska, M., and Silver, I.A. 1991. Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am. J. Physiol. 260: C1131–C1150. Wallace, J.C. 1973. Feeding, starvation and metabolic rate in the shore crab Carcinus maenas. Mar. Biol. (Berl.), 20: 277–281.

© 1999 NRC Canada

J:\cjz\cjz77\cjz-05\Z99-021.vp Friday, October 01, 1999 2:34:03 PM