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Mechanism for maintaining oxygen consumption under varying oxygenation levels in the freshwater clam Corbicula fluminea Damien Tran, Alain Boudou, and Jean-Charles Massabuau

Abstract: The basic adaptation mechanisms that allow the Asian freshwater clam Corbicula fluminea to maintain its oxygen (O2) consumption constant under resting conditions when the partial pressure of O2 (PO 2 ) in the water varies from 4 to 40 kPa were studied at plankton concentrations which were high enough that ventilation was not affected. Steady-state values for O2 consumption, PO 2 , and O2 concentration in the arterial and venous blood, PO 2 in the expired water, and ventilatory and circulatory blood flow were determined after 24-h periods of exposure to selected water PO 2 values. The key adaptation after 1 day of acclimation was the maintenance of O2 consumption, which was achieved exclusively by ventilatory adjustment, with no change in the oxygenation status of the internal milieu. Specifically, arterial PO 2 remained constant at 3 kPa and venous PO 2 at 2 kPa. Arterial and venous blood O2 concentrations and bloodflow rate also remained constant. The data are discussed in terms of feeding versus respiratory control of ventilation in filter-feeders in an environmental context. The agreement between the homeostasis strategy described here and previous results reported for the freshwater mussel Anodonta cygnea, crustaceans, and teleosts is emphasised. 2036 Résumé : Les principes de base qui permettent au mollusque Corbicula fluminea de maintenir sa consommation d’oxygène quand la pression partielle d’oxygène, PO 2 , varie dans l’eau de 4 à 40 kPa ont été étudiés dans des conditions où la concentration de plancton était suffisamment élevée pour ne pas stimuler la ventilation. La consommation d’oxygène, la pression et la concentration d’O2 dans les sangs artériels et veineux, PO 2 dans l’eau expirée, les débits ventilatoires et circulatoires ont été déterminés à l’état d’équilibre après 24 h d’exposition à différents niveaux d’oxygénation dans l’eau. L’adaptation clef après 1 jour d’exposition est le maintien de la consommation d’oxygène réalisé exclusivement par un ajustement de l’activité ventilatoire sans changement de l’état d’oxygénation du sang. Spécifiquement, PO 2 dans le sang artériel est maintenu constant à 3 kPa et dans le sang veineux à 2 kPa; la concentration d’O2 dans les sangs artériels et veineux ainsi que le débit sanguin restent aussi constants. Les résultats sont discutés, dans un contexte environnemental, en terme de contrôle de la ventilation orientée vers la fonction respiratoire ou nutritive chez un filtreur. L’analogie avec des résultats obtenus précédemment chez le mollusque Anodonta cygnea, des crustacés et des poissons téléostéens est mise en avant.

Introduction The freshwater Asian clam Corbicula fluminea is an invasive species that occurs naturally on several continents (Asia (including New Guinea), Africa, and Australia; Zadhin 1965). It was introduced into North America sometime prior to 1938, and it is now widely distributed south of latitude 40°N (Britton and Morton 1982). Since the 1970s it has invaded western Europe (Mouthon 1981). Its presence in North America raises some very important ecological and economic problems, as its density is great enough to constitute a real challenge in many bodies of water. Based on its wide geographical distribution and continuous water-filtering activity, it is often used for aquatic toxicology because of its potential to accumulate various water contaminants (Hemelraad Received January 27, 2000. Accepted July 27, 2000. D. Tran, A. Boudou, and J.-C. Massabuau.1 Laboratoire d’Ecophysiologie et Ecotoxicologie des Systèmes Aquatiques, University Bordeaux 1, and Unité mixte de recherche no 5805, Centre National de la Recherche Scientifique, Place du Docteur Peyneau 33120, Arcachon, France. 1

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

Can. J. Zool. 78: 2027–2036 (2000)

et al. 1988; Cassani et al. 1986; Baudrimont et al. Tran et 1986, al. 1997). In the field of instantaneous biomonitoring, a number of attempts have been made to use molluscs as sentinel organisms, as they are able to reveal changes in water quality rapidly through modifications of their valve activity (Doherty et al. 1987; Salanki et al. 1991; Borcherding 1994; Ham and Peterson 1994). However, despite a large amount of data concerning both mollusc physiology and the above fields of interest that are based either directly or indirectly on ventilatory activity, few works have been devoted specifically to their ventilation-control mechanisms. Since the pioneering work of Van Dam (1938) and Krogh (1941), numerous authors have concluded that ventilation in filter-feeding organisms is maintained far beyond their respiratory requirements for feeding purposes and that the respiratory function is incidental. Recently, despite some studies indicating that a bivalve can adjust its ventilation to achieve some “optimal” rate of particle retention (Hornbach et al. 1984; Foe and Knight 1986; Way et al. 1990), Jorgensen (1990) proposed that in molluscs there is no physiological control of feeding or respiration rates through the regulation of pumping rates. When analysed from a truly respiratory standpoint (Dejours 1981), this possible absence of ventilatory control raises very specific problems. Indeed, the existence of a global strategy © 2000 NRC Canada

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Can. J. Zool. Vol. 78, 2000 spired by the clam). The animals were put in place 24 h prior to any measurements being taken (time t0), and measurements of M& O 2 were systematically started the next morning from 10:00 a.m. to 12:00 noon (t0 + 23–24 h) in normoxia. Each animal was exposed to 6 selected and maintained water PO 2 levels (40, 20, 4, 3, 2, and 1 kPa). The order of presentation was 20, 40, 4, 3, 2, 1 to prevent an eventual O2-debt repayment in hypoxia. The duration of exposure was 24 h and the animals were allowed to adapt to each water PO 2 plateau for 22 h, after which measurements were performed. Water samples entering and exiting the respirometer were withdrawn through stainless-steel tubes into glass syringes at a rate of 150 µL·min-1 for 10 min using a Perfusor pump (Braun Melsungen). Oxygen consumption by bacteria contaminating the collection system was undetectable. PO 2 was measured with a Radiometer polarographic electrode at 15°C. The N2/O2/CO2 gas mixture was obtained via a laboratory-constructed mixing unit (rotameters and valves from the Brooks Company). The Fick principle was used to calculate M& O 2 when the chamber–animal system reached steady state:

of adaptation based on the maintenance, mainly by ventilatory adjustments, of a low and constant oxygenation level in the internal milieu independently of the ambient oxygenation level, has been largely documented in the other two major groups of aquatic animals, crustaceans and fishes (Massabuau and Burtin 1984; Forgue et al. 1989; Takeda 1990; Legeay and Massabuau 1999). In this study we addressed the problem of determining whether there is a mechanism that regulates the oxygen (O2) supply in C. fluminea exposed to various O2 partial pressures (PO2 ) at plankton concentrations which were high enough that ventilatory activity was not affected. In particular, care was taken to reduce the external stimulation usually associated with laboratory experiments to a minimum by using isolated experimental units placed on antivibration benches. We show that C. fluminea is able to maintain its steady-state O2 consumption constant at water PO2 values ranging from 40 to 4 kPa (20–2 mg·L–1) exclusively by means of ventilatory adjustments and with no change in blood-flow rate. Its strategy is based principally on maintaining the oxygenation of its internal milieu within a low and narrow range. By analogy, between the blood parameters measured in this study and those known from other animals (Massabuau and Burtin 1984; Bouverot 1985), it is suggested that arterial PO2 , Pa O2 , can be a controlled variable in fed molluscs.

where M& O 2 is O2 consumption (µmol·h–1·g-1 FM); V&r is the waterflow rate through the respirometer (mL·min–1); αw O 2 is the O2 solubility coefficient (15.08 µmol·L–1·kPa–1); Pi O 2 is the PO 2 in water entering the respirometer (kPa); and Po O 2 is the PO 2 in water leaving the respirometer (kPa).

Material and methods

Blood-oxygenation status at various water-oxygenation levels

Animals and ambient conditions Experiments were performed on 100 C. fluminea weighing 0.88– 5.07 g fresh body mass, shell excluded (FM), and measuring 30.2 ± 0.7 mm from the anterior to the posterior aspect of the shell valves. The bivalves were collected locally and kept in large tanks supplied with tap water under the following conditions: temperature 15.0 ± 0.5°C; PO 2 20–21 kPa; pH ≈ 7.80 ± 0.10; and water titration alkalinity 1.85 ± 0.10 mmol·L–1. The bottom of the tanks was covered with a 1:1 mixture of quartz sand (Silaq France, granulometry 0.8– 1.4 mm) and natural sediment from the River Garonne. The animals were fed twice a week with algae (Scenedesmus subspicatus). Before any experiments were undertaken, the animals were acclimated in the laboratory for at least 3 weeks to settle their ventilatory activity. Experiments were carried out throughout the year under natural light conditions, and during experiments the tanks were isolated from laboratory vibrations through the use of antivibration benches to minimise external disturbance. For reference, 1 kPa = 7.5 Torr or mmHg, and in water, PO 2 = 1 kPa corresponds to an O2 fraction ≈1% and an O2 concentration ≈0.5 mg·L–1 or ≈0.3 mL·L–1 at 15°C (in water equilibrated with air, i.e., “normoxic,” the O2 fraction is 21% and PO 2 ≈ 21 kPa). Three types of experiments were performed at various steady-state water PO 2 levels: (1) O2-consumption measurements; (2) bloodoxygenation analysis; (3) determination of ventilatory activity, bloodflow rate, and PO 2 in the expired water.

Experimental protocol Measurement of O2 consumption Experiments were performed on 12 C. fluminea. Oxygen consumption, M& O 2 (µmol·min–1·kg–1), was measured in an open-flow respirometer, volume 60 mL (water-flow rate 0.62 mL·min-1), using the technique described by Massabuau et al. (1984). The main feature was a rotor that ensured a homogeneous composition within the system (the result being that the water exiting the chamber, the composition of which is controlled, closely resembled the water in-

[1]

& M& O 2 = Vr·αw O 2 (Pi O 2 – Po O 2 )

This experiment was performed on 81 C. fluminea. The protocol of the PO 2 plateau exposures previously described was repeated and at each oxygenation level, PO 2 was measured in the arterialised blood in the heart and in the venous blood sampled from the venous lacunae of the posterior adductor muscle. Animals were fed every 2 days.

Experimental set-up and blood sampling—Preliminary experiments on C. fluminea showed that, as already reported for the freshwater mussel Anodonta cygnea (Massabuau et al. 1991), stress is an especially difficult problem to handle in molluscs. Indeed, when we first started to sample a group of C. fluminea resting in a single tank (20 L) for a few days, we recorded a clear increase in arterial PO 2 as a function of sampling order. Therefore, to circumvent this artefact, each animal was kept in a single 100-mL chamber individually supplied via gravity with water at constant flow (400 mL·h–1). Each chamber (n = 13–14) was individually isolated on a sand bed and half-filled with quartz sand. The gas mixtures were bubbled through the reservoir of water supplying the chambers. For blood sampling, animals were prepared at least 10 days prior to the experiments, and in two series, one for venous sampling and the other for arterial sampling. Figure 1 shows how the clams were prepared (see insets), as well as the blood-sampling locations. A deep groove was drilled into the shell either below the posterior adductor muscle (Fig. 1A, inset) or above the heart (Fig. 1B, inset) to weaken the shell. Blood samples were collected by gently removing the animals from the water, instantaneously breaking the shell at the desired level with a modified oyster knife, and, under a binocular microscope and using a glass capillary tube, puncturing (i) the heart for arterial sampling or (ii) the posterior adductor muscle for venous sampling. All sampling was performed between 10:00 a.m. and 4.00 p.m. Arterial (50 µL) and venous (80 µL) blood samples were obtained within the first 60–90 and 30–45 s of emersion, respectively. This sampling technique, which is associated with emersion, was critically assessed in Forgue et al. (1992) and Massabuau and Forgue (1996) in relation to crustaceans and compared favourably with the use of the chronic catheterisation. © 2000 NRC Canada

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Fig. 1. Arterial and venous blood sampling in the Asian clam Corbicula fluminea. Prior to any sampling, a deep groove was drilled in the shell to weaken it (see insets). (A) Venous blood sampling from the posterior adductor muscle. (B) Arterial blood sampling by puncturing the heart.

Blood-gas analysis—After sampling, the glass capillary tubes were plugged and blood PO 2 was determined within 3 min on 50-µL samples with an E5046 Radiometer polarographic electrode housed in a laboratory-modified electrode chamber thermostatted at 15°C. The electrode was calibrated with a zero PO 2 solution (S4150 Radiometer) and air-equilibrated fresh water. Before measurements, low PO 2 equilibrated water was systematically injected into the electrode to decrease its response time and improve its accuracy in the low range. As shown in Massabuau and Forgue (1996, Fig. 2), the present calibration procedure, which improves analysis quality in the low range, does not preclude the reading of high blood PO 2 values. As the blood of C. fluminea lacks any respiratory pigment, the O2 concentration in the arterial and venous blood (Ca O 2 and CvO 2 , respectively, µmol·L–1) was calculated according to Henry’s law (C O 2 = α O 2 ·PO 2 ), using a solubility coefficient, α O 2 , of 15.3 µmol·L–1·kPa–1 for water at 15°C. The O2 concentrations were & then compared with the M& O 2 values, and the blood-flow rate, Vw (mL·h–1·g–1 FM), was estimated using the Fick principle (see Dejours 1981). [2]

& M& O 2 = Vb·αb O 2 (Ca O 2 – Cv O 2 )

where M& O 2 is O2 consumption (µmol·h–1·g–1 FM); αb O 2 is the O2 solubility coefficient of the blood; Ca O 2 and CvO 2 are the O2 concentration in the arterial and venous blood (µmol·L–1), respectively. The corresponding coefficient of O2 extraction from the blood, Eb O 2 (%), was estimated as [3]

Eb O 2 =

(Ca O 2 – CvO 2 ) Ca O 2

× 100

Measurement of ventilatory flow rate This measurement, based on determination of the volume of water cleared of algae per unit of time in a temporarily closed system (Coughlan 1969; Jorgensen 1990), was performed on 7 animals held separately in individual chambers (volume 100 mL) half-filled with quartz sand. We repeated the protocol used for the 24-h PO 2 plateau exposures previously described, and calculated, for each oxygenation level, the individual water-flow rate for each clam in steady state. Importantly, the present protocol was characterised by (i) maintaining the clams in open-flow systems held at constant

water PO 2 and alga concentration (±0.5 × 105 of the nominal value in the range 2–12 × 105 algae/mL) for 22 h prior to analysis, and (ii) confining them during the 1-h measurement periods. In the open-flow systems, each chamber was constantly supplied with pre-oxygenated water at constant flow (400 mL·h–1) via gravity and the alga (S. subspicatus) solution via a water-renewal pump (Gilson). The gas mixture was bubbled through the reservoir of water supplying the chambers, then at a low rate into each chamber, where it improved water homogenisation and gas equilibration throughout the experiment, especially during the periods of confinement. During these periods, water flow through the chamber was stopped and, at constant water PO 2 , the actual alga concentration decreased from 1 to 3 × 105 algae/mL, depending on the reference concentration, the water PO 2 , and the ventilatory activity of each individual. Alga density was measured as optical density at 750 nm with a spectrophotometer, UV-1601 Shimadzu, regularly calibrated by counting cells with a Malassey cell, using light microscopy. The clams were placed in the experimental set-up at least 3–4 days before the experiment began, and analyses were always performed between 11:00 a.m. and 4:00 p.m. Jorgensen’s & the equation (cited in Coughlan 1969) was used to calculate Vw, ventilatory flow rate of the clams: [4]

& = V [ ln(do) – ln(df)] − V [ ln(do′ ) − ln(df′ )] Vw ( tM ) ( tM )

& is the ventilatory flow rate (mL·h–1·g–1 FM); V is the where Vw volume of water in each chamber (mL); ln(do) and ln(df) are the natural logarithm of alga density (algae/mL) at the beginning and end of the measurement period, respectively; ln(do′ ) and ln(df′ ) are the control alga density (algae/mL) in the control chamber without clams, at the beginning and end of the measurement period, respectively; t is the duration of each exposure period, which was consistently ≈60 min; and M is body mass (FM; g). It is important to note that four assumptions are associated with the use of eq. 4: the concentration of algae in the water was homogeneous as measured in the control experiment; the decrease in alga concentration was due only to animal filtration, as confirmed by the lack of change within the empty control chamber; all the algae passing over the gills were retained; and the ventilatory flow rate was constant throughout the measurement period. © 2000 NRC Canada

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Can. J. Zool. Vol. 78, 2000 Table 1. Respiratory variables (mean ± SE) measured in the Asian clam Corbicula fluminea exposed to selected fixed oxygenation levels in the water (T = 15°C). PI O 2 (kPa) M& O 2 (µmol·h–1·g–1) & (mL·h–1·g–1) Vw –1 –1 –1 & Vw·αw O 2 (µmol·kPa ·h ·g )* & (mL·h–1·g–1)* Vb –1 –1 –1 & Vb·αa,v O 2 (µmol·kPa ·h ·g )* & V&b–1* Vw· & M& O –1 (mL·µmol–1)* Vw· 2 & M& O –1 (mL·µmol–1)* Vb· 2 PE O 2 (kPa)* )PI,E O 2 (kPa)* Ew O 2 (%)* Pa O 2 (kPa) Pv O 2 (kPa) Ca O 2 (µmol·L–1)* Cv O 2 (µmol·L–1)* )Ca,v O 2 (µmol·L–1)* Eb O 2 (%)*

4.3±0.1

19.9±0.2

38.8±0.3

0.17±0.03 64.9±9.9 0.99±0.15 9.7±0.4 0.15±0.01 6.7 381.8 57.1 4.1±0.1 0.2 4.1±0.5 3.1±0.2 1.9±0.1 48.4±3.1 29.7±1.6 18.7 38.7

0.19±0.02 15.7±4.0 0.24±0.06 9.1±0.5 0.14±0.01 1.7 82.6 47.9 19.1±0.3 0.8 4.1±0.5 3.4±0.3 2.1±0.2 53.1±4.7 32.8±3.1 20.3 38.2

0.19±0.04 6.4±1.5 0.10±0.02 7.1±1.5 0.11±0.02 0.9 33.7 37.4 36.9±0.6 1.0 4.9±1.0 4.2±0.7 2.4±0.3 65.6±10.9 37.5±4.7 28.8 42.8

Note: Values without an asterisk were directly measured and those with an asterisk were calculated. PI O 2 , O2 partial pressure in the inspired water; M& O 2 , O2 consumption per unit of fresh body mass (FM); V& w, ventilatory-flow rate per unit of body mass; V& w·"w O 2 , ventilatory conductance per unit of body mass; V& b, circulatory blood flow rate per unit of body mass; V& b·αa,v O 2 , perfusive conductance per unit of body mass; V& w·V& b–1, ventilation/perfusion ratio; V& w·M& O 2 –1, specific ventilation rate (volume of water breathed in order to extract a unit quantity of O2); V& b·M& O –1, specific circulation rate; PE O , P O in the 2

2

2

expired water; )PI,E O 2 , difference in P O 2 between the inspired and expired water; Ew O 2 , extraction coefficient of O2 from water; Pa O 2 , P O 2 in the arterial blood; Pv O 2 , P O 2 in the venous blood; Ca O 2 , O2 concentration in the arterial blood; Cv O 2 , O2 concentration in the venous blood; )Ca,v O 2 , difference in CO2 between arterial and venous blood; Eb O 2 , extraction coefficient of blood O2. Mean body mass was 3.27 ± 1.62 g FM (range 0.88–5.07 g FM); n = 6–14 animals per data point (see the text). All variables are from Dejours (1981).

Expired water—Based on the O2-consumption and ventilatoryflow rates measured under the same experimental conditions and at the same PO 2 values for the inspired water (PI O 2 ), the Fick principle was used to estimate mean PO 2 values for the expired water (PE O 2 ): [5]

& ⋅ αw O (PI O – PE O ) M& O 2 = Vw 2 2 2

& and αw O have their usual meaning, PI O is the PO where Vw 2 2 2 value of the inspired water and PE O 2 is the P O 2 of the expired water. The extraction coefficient of O2 from water, Ew O 2 , was calculated according to Dejours (1981) on paired P O 2 values of the inspired and expired water: [6]

Ew O 2 =

( PI O 2 − PE O 2 ) PI O 2

× 100

Statistics The results are given as frequency-distribution plots of means ± 1 SE and (or) trimmed means (Winsorised estimator) to estimate modal values in the case of rare occurrences of extreme values (Lecoutre and Trassi 1987). Differences were evaluated using analysis of variance (ANOVA, one-way), Mann–Whitney and Wilcoxon’s tests, and Fisher’s LSD method. P < 0.05 was taken as the fiducial limit of significance.

Results Table 1 shows all the respiratory variables measured and calculated for C. fluminea acclimated to conditions in our laboratory and exposed to selected and fixed water PO2 values at 15°C. Figure 2 illustrates their ability to maintain their O2 consumption constant in the water PO2 range 40– 2 kPa (n = 6–12). Only below a water PO2 of 2 kPa was M& O2 significantly lowered, although the animals were nonetheless able to maintain their reduced M& O2 for 24 h and to recover when they were replaced in normoxic conditions. This sheds new light on the adaptive potential of the species and clearly shows that it is, at least when resting, an excellent “O2 regulator.” We next addressed the issue of the underlying mechanisms associated with this maintenance and turned to blood-gas analysis for insights into the oxygenation status of the internal milieu. The arterial and venous PO2 values for C. fluminea exposed at a water PO2 of 4, 20, or 40 kPa (n = 13–14 clams per water-oxygenation level) during 24-h exposure periods are presented in Fig. 3. These were characterised mainly by being low, regardless of the water PO2 . The most frequently measured arterial PO2 (Pa O2 ) values were in the range 2–4 kPa (with only one exceptional value of 12 kPa © 2000 NRC Canada

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Fig. 2. Relationship between oxygen (O2) consumption and water-oxygenation level in C. fluminea in resting conditions. Steady-state values were measured after 22–24 h of acclimation. Oxygen consumption remained constant down to PI O 2 = 2 kPa (n = 6–12 animals per data point). Values are given as the mean ± 1 SE (*, significantly different from normoxia; P < 0.05, Wilcoxon’s test, one-way ANOVA, and Fisher’s LSD method).

in hyperoxia) and the venous PO2 (Pv O2 ) values were mostly between 1 and 3 kPa. This consistency is typical of the resting state, as already reported in numerous other physiologically different water-breathers (see the Introduction). Figure 4A shows the corresponding change in arterial and venous O2 concentrations in this bivalve that lacks any blood respiratory pigment. As there was a direct relationship between blood PO2 and blood O2 concentration, the latter also remained in a steady range, regardless of water PO2 . The evolution of the calculated blood-flow rate is presented in Fig. 4B, which again shows remarkable consistency. Finally, note that in Fig. 4C, the extraction coefficient of O2 from blood reached about 40% regardless of the water PO2 within the range studied. Thus, taken together these data illustrate a remarkable strategy of homeostasis in the internal milieu in terms of O2. We then focused our attention on the potential existence of a O2 ventilatory drive. The change in ventilatory flow rate determined in 7 C. fluminea exposed to 4 selected and maintained water PO2 levels is shown in Fig. 5. It is clear that the level of ventilatory activity was inversely related to the wateroxygenation level. Note that at a water PO2 of 4 kPa, the ventilatory flow rate was 10 times greater that in hyperoxia, 40 kPa (64.9 ± 9.9 versus 6.4 ± 1.4 mL·h-1·g-1 FM). Finally, note also that in the present resting conditions, the ventilatory flow rate in normoxia was 15.7 ± 4.0 mL·h–1·g–1 FM, which is less than 400 mL·d–1·g–1 FM. As the method we used to indirectly calculate the rate at which C. fluminea processes water was consistently associated with a decrease in alga concentration, we then studied the extent to which this could influence ventilatory activity. Consequently, we tested the effect of short-term decreases in alga concentration at various concentrations ranging from 2 to 12 × 105 algae/mL and various water PO2 levels. Figure 6 shows the relationship between ventilatory flow rate and alga density at 15°C for 3 water-oxygenation levels. It is clear that in the range studied, ventilatory activity was independent of alga density, and was only dependent on the water PO2 . Finally, the PE O2 values and the coefficient of O2 extraction from water were calculated for various PI O2 values. The results are presented in Fig. 7, and show that PE O2 always remained remarkably high and very close to PI O2 (Fig. 7A), which corresponds to an extremely low and constant coefficient

Fig. 3. Frequency distributions of arterial and venous O2 partial pressures in resting C. fluminea. Steady-state values were measured after 22–24 h of acclimation (PI O 2 = 4, 20, and 40 kPa). Distributions are not normal. The most frequently measured partial pressure of O2 (P O 2 ) values were in the range 1–3 kPa in the arterial blood and 1–2 kPa in the venous blood (n = 13–14 animals per distribution; see Table 1 for mean arithmetical values). The vertical dotted line represents 3 kPa.

of O2 extraction from the ventilated water, 4%, independent of PI O2 (Fig. 7B).

Discussion In this study we present evidence that in C. fluminea, (i) a strong ventilatory O2 drive does exist; (ii) at least in resting and fed conditions, ventilation appears to be adjusted so that © 2000 NRC Canada

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Fig. 4. Relationships between steady-state values of blood O2 concentration in the arterial and venous blood (A), blood flow rate (B), and coefficient of blood O2 extraction, Eb O 2 (C), as a function of PI O 2 in C. fluminea. All values remained independent of PI O 2 (n = 13–14 animals per data point). Values are given as the mean ± 1 SE (P < 0.05, one-way ANOVA and Fisher’s LSD method).

Fig. 5. Ventilatory flow rates as a function of PI O 2 in C. fluminea. Steady-state values were measured after 22–24 h of acclimation. The ventilatory flow rate is inversely related to the water-oxygenation level. Values with an asterisk are significantly different from the reference value in normoxia (P < 0.05, Wilcoxon’s test, one-way ANOVA, and Fisher’s LSD method; n = 7 animals per data point). Values are given as the mean ± 1 SE.

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Fig. 6. Relationship between ventilatory-flow rate and plankton concentration, ranging from 2 to 12 × 105 algae/mL, at various PI O 2 values (4, 20, and 40 kPa) for C. fluminea. Data are presented as individual values for 7 animals (each type of symbol denotes an individual animal). (A) Three measurements were made per animal, at various alga concentrations. (B and C) Eight measurements were made per animal. The ventilatory-flow rate is independent of the plankton concentration in the range studied (P < 0.05, one-way ANOVA and Fisher’s LSD method).

Fig. 7. Relationship between PE O 2 , PI O 2 in the expired water (A), and Ew O 2 , the extraction coefficient of O2 from the water (B), as a function of PI O 2 , P O 2 in the inspired water, in C. fluminea. The extraction coefficient of O2 from the ventilated water remained in the 4–5% range whatever the PI O 2 value (n = 13–14 animals per data point; P < 0.05, Mann–Whitney test, one-way ANOVA, and Fisher’s LSD method).

in terms of O2, homeostasis of the internal milieu remains remarkably constant whatever the water P O2 level between 40 and 2 kPa (the ventilatory-flow rate was inversely related to the water-oxygenation level), and (iii) this is performed without any change in blood (arterial and venous) P O2 , O2 concentration, or flow rate. In contrast, a comparison with ventilatory adjustments associated with food uptake shows that at 15°C, a concentration of monospecific plankton (S. subspicatus) in the range 2–12 × 105 algae/mL does not influence ventilatory activity. Thus, in terms of ventilationcontrol mechanisms, we propose that C. fluminea, like numerous other physio- logically different water-breathers including fishes and crustaceans, has a strategy for maintaining resting aerobic metabolism in resting conditions, which, by

analogy with previous reports (Massabuau and Burtin 1984; Forgue et al. 1989; Bouverot 1985), is based principally on ventilatory control of Pa O2 . Since, in terms of O2, it has been reported A. cygnea provides a similar example of homeostasis of the internal milieu (Massabuau et al. 1991), we suggest that for molluscs such a strategy could have some general value. In these animals the change in ventilatory flow rate could theoretically be based on either a change in ventilation rate over the gills or a change in the duration of valve opening/closing. In our experimental conditions, the proportion of intervals when the valves were closed during daytime was consistently low, as previously reported by Ham and Peterson (1994) for the same species. Consequently, we suggest that C. fluminea can maintain its © 2000 NRC Canada

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O2 consumption by continuously adjusting its ventilatory activity. The effect of changing water-oxygenation levels on ventilation and O2 consumption has often been studied in molluscs, but the control mechanisms that allow O2 consumption to be kept constant at various water P O2 levels have received little attention. The steady-state O2 consumption that we measured in normoxia (0.10 µmol·h–1·g–1 FM) was comparable to previous data (the dog cockle, Glyceremis glyceremis, Brand and Morris 1984; A. cygnea, Massabuau et al. 1991), but remained in the lower part of the range of most reported values. Specifically, it was significantly lower than the 1.2– 3.9 µmol·h–1·g–1 FM reported by McMahon (1979) in normoxic C. fluminea at 10 and 20°C, respectively. Although the experimental approach was different (McMahon (1979) used a closed respirometer, whereas we used an open-flow system), this lack of agreement clearly requires clarification. The ability to maintain O2 consumption constant when water oxygenation decreases has been regularly reported in bivalves but never in C. fluminea, which, to our knowledge, has always been described as an oxyconformer (Aldridge and McMahon 1978; Lenat and Weiss 1973; McMahon 1979). It has been shown, for example, that M& O2 can be maintained constant in Mytilus perna L. down to ≈10 kPa at 20°C (Bayne 1967), in G. glyceremis, down to 2.5 kPa at an unknown temperature (Brand and Morris 1984), in Pleurobema coccineum down to 0.7–2 kPa at 20°C (Badman 1974) and in A. cygnea down to 0.9 kPa at 13°C (Massabuau et al. 1991). The blood-flow rates we measured were in the range of the literature data (b = 13–36 mL·h–1·g1 FM in Modiolus demissus and 1.5 mL·h–1·g1 FM in Noetia ponderosa; Booth and Mangum 1978). Similarly, the coefficient of blood O2 extraction that we calculated (38%) fits well within the 47– 14% range measured in N. ponderosa and M. demissus (Booth and Mangum 1978). Finally, note that our report fits well with the observations of Johnson and McMahon (1998), who studied the hypoxia tolerance of C. fluminea and Dreissena polymorpha. They demonstrated that at 15°C, C. fluminea can successfully face exposure to a water PO2 of 1.6 kPa for about 10 days without mortality. In the literature on molluscs, our finding that blood oxygenation can remain in a low and narrow range in a bivalve, regardless of the water PO2 (Figs. 2B, 2C), is consistent only with the results of our own previous study on A. cygnea (Massabuau et al. 1991). Two other major findings in the present study are, first, the fundamental and unique role played by ventilatory adjustments and second, the systemati& < cally low ventilatory flow rate measured (in normoxia, Vw –1 –1 0.4 L·d ·g FM). We suggest that apart from possible speciesspecific factors, this can largely be explained by the particular protocols we used to isolate the experimental animal from laboratory stimulation. Nevertheless, although comparisons with numerous literature data were difficult, owing to the use of the particular protocol plus different planktonic particle types, temperature, acclimation periods, etc., the present ventilation measurements are obviously in the low range of most reported values for C. fluminea and molluscs in general. Specifically, they are quite close to the results of Prokopovitch (1969), obtained at 20°C, who measured 0.48 L·d–1·g–1 FM in C. fluminea, but quite different from

Can. J. Zool. Vol. 78, 2000

the data of Foe and Knight (1986), who reported ≈7.2– 9.6 L·d–1·g–1 FM at 20°C for plankton concentrations ranging from 1 to 10 × 105 algae/mL. In terms of comparison with other molluscs, it has been reported that the mussel M. edulis ventilates 41 L·d–1 (T = 12–15°C, shell length 48– 80 mm; Willemsen 1952) and the oyster Crassostrea gigas, 250 L·d–1·g–1 FM (Jorgensen 1975). This range obviously covers most of the values that can be measured in bivalves. Whether or not the present analysis relating to C. fluminea applies to a specific field situation remains to be studied. Despite the low ventilatory flow rates observed in the present study, and the corresponding long water-transit times in the branchial and palleal cavities, the coefficients of O2 extraction from water appeared to be quite low in C. fluminea. Very few data are available to allow a comparison (except in M. demissus, where Ew O2 = 8–9% (Booth and Mangum 1978), and A. cygnea, where Ew O2 = 2–10% (Van Dam 1938)), but this raises the very specific problem of O2-diffusion limitation. Indeed, in C. fluminea, at least at the highest water P O2 , the present results show that there is no P O2 equilibrium between PI O2 and Pa O2 nor between PE O2 and Pv O2 . Based on the theoretical approach developed by Piiper and Scheid (1984), this suggests that diffusion limitation must be important in this gas exchanger and that O2 exchanges should be ventilation-limited. The functional basis of this limitation is attributed to a mismatch between ventilation and perfusion at all water P O2 levels. The magnitude of this shunt can be estimated from the fraction PI O2 – Pa O2 / PI O2 – Pv O2 , which is the scale of the mismatch compared with the difference between PI O2 and Pv O2 taken as a reference. In normoxia the size of the shunt was 0.95, which is remarkably large. It decreases in hypoxia to 0.5 at a water P O2 of 4 kPa. Two alternative but not necessarily mutually exclusive explanations are possible. The first is that the decrease in the shunt fraction in hypoxia may correspond to an increase in the ventilated gill area or in its blood-perfusion rate. Alternatively, our data may also indicate that a significant diffusion barrier and (or) unstirred layer exists in C. fluminea gills at a low ventilatory-flow rate. In fact, both could contribute significantly in the absence of an O2 equilibrium between blood and water, whatever the animal’s ventilatory-flow rate. To our knowledge, no study has specifically addressed the problem of O2 diffusive resistance in molluscan gills, although a large amount of work has been devoted to gill anatomy (see Morse and Zardus 1997). In C. fluminea, when only epithelium thickness is considered, the water–blood distance (Lemaire-Gony and Boudou 1997) is comparable to that generally found in other water-breathers in which the mean water–blood barrier varies from 0.5 to 10 µm (Taylor and Taylor 1992; Hughes 1984). An important point, however, is the presence of mucocytes in the respiratory epithelium, as their role is difficult to determine. In our opinion, they could have a significant impact regarding O2-diffusion problems, as they act as a diffusion barrier limiting gas exchange. A further question is the probably not negligible local consumption of O2 used to fuel the cilia, which could limit the net O2 transfer from water to blood. Global blood O2 uptake appears, then, to be the result of quite a complicated balance; nevertheless, and contrary to previous proposals (Krogh 1941; Jorgensen 1990), it is obvi© 2000 NRC Canada

Tran et al.

ously the end-product of a well-defined regulatory process in which Pa O2 appears to be a major controlled variable that can be entirely maintained by means of ventilatory adjustments. In the resting C. fluminea studied in our experimental conditions it is clear that this cannot be the passive result of ventilatory activity constantly maintained for feeding purposes. The existence of a relationship between ventilatory activity and water PO2 has already been observed in G. glyceremis by Brand and Morris (1984), who reported that the ventilatoryflow rate was inversely related to water oxygenation. The present results demonstrate the existence of a strong O2-ventilation drive in C. fluminea but not the absence (or presence) of any drive associated with feeding. They simply suggest that at 15°C, during daytime and in resting conditions, the concentrations of plankton we used were always sufficient to ensure proper food uptake, even when ventilation was slowed down by the excess O2 in hyperoxia. It is very likely that a ventilatory drive associated with feeding would appear at plankton concentrations lower than 2.105 algae/mL and (or) at higher metabolic levels, when, for example, the temperature increases. But in this respect, and in an ecological context, it must be kept in mind that the present experimental temperature is fairly representative of the mean annual value that can be observed in numerous continental climates where water temperatures higher than 20–25°C are rare and restricted to a few weeks in summer (Anonymous 1999; Massabuau and Fritz 1984). Similarly, the concentrations of the monospecific plankton (S. subspicatus) that we used, which varied in the range 2–12 × 105 algae/mL (equivalent to 18–56 µg chlorophyll a·L–l) fit well with numerous field conditions (OCDE 1982). Consequently, the present results, as they stand, strongly suggest that during the time period when the field temperature is ≤15°C, i.e., during most of the year in many temperate and continental climatic areas, in numerous water bodies a low ventilation rate & = 400 mL·g–1·d–1 in normoxia) is enough in C. fluminea (Vw to satisfy feeding requirements. Finally, one must keep in mind the fact that the water P O2 is extremely variable in the natural environment and that C. fluminea inhabit very different biotopes, although they are restricted to locations where the water column is systematically well oxygenated (Johnson and McMahon 1998). On the one hand, Britton and Morton (1982) reported populations living burrowed in the soft silts of the deeper waters of lakes, evidently with the inhalant siphon a few millimetres above the substrate, where the water P O2 at the interface can be low. On the other hand, there are populations living in rivers with considerable photosynthetic activity. As was stressed earlier, at one of our sampling sites we measured a water P O2 of 36 kPa on a sunny afternoon in May. Adapting to these wide variations in wateroxygenation level could therefore be an important ventilatory drive for C. fluminea in the field. In conclusion, the present results demonstrate the existence of a sizeable O2-ventilation drive in C. fluminea in conditions where the ventilation rate is enough to satisfy food-uptake requirements, regardless of the plankton concentration. This was associated with maintaining the O2 homeostasis of the internal milieu within a low and narrow range. This strategy, which can have very profound physiological consequences (Massabuau and Meyrand 1996; Clem-

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ens et al. 1998 1999), thus appears increasingly likely to be a basic property of respiratory physiology in resting waterbreathers, even those in which ventilation serves for both respiration and feeding.

Acknowledgements The authors thank Dr. Gilles Durrieu for his critical reading of the manuscript. D.T. was supported by a grant from the French Ministry of Research and Education. All the experiments presented in this paper complied with the current laws of France, where they were performed.

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