Low Arterial in Resting Crustaceans is Independent of Blood Oxygen-Affinity

Abstract

In the literature about the respiratory physiology of water-breathers, the arterial partial pressure of oxygen, , is reported to range from about 2 to 10–13 kPa (Shelton et al. 1986; McMahon and Wilkens, 1983). In fish, Shelton et al. (1986) reported that animals with low blood O2-affinity exhibit high and vice versa. But in laboratory conditions, the excitation state of the animals may explain much of this variability: high excitation is associated with high values and low excitation with low , values (see McMahon, 1985, for relevant discussion on crustaceans). In conditions where animal excitability was reduced to a minimum, we reported that low values correspond to an apparent set point in crayfish Astacus leptodactylus (Massabuau and Burtin, 1984), fish Silurus glanis (Forgue et al. 1989) and mussel Anodonta cygnea (Massabuau et al. 1991). Indeed, when the inspired varies from 3 to 40 kPa, remains in the range of 1–3 kPa. In resting crabs Carcinus maenas - contrary to all previously reported data (see McMahon and Wilkens, 1983) - and Eriocheir sinensis, we also reported that in normoxic conditions is kept in a low range around 1–3 kPa. This is independent of the blood pigment concentration (Forgue et al. 1992).Except for A. cygnea, which has no respiratory pigment, the above species exhibit mid values of blood O2-affinity, with P50 (the values of at which 50% of the respiratory pigment is oxygenated) around 0.7-1 kPa at physiological pH (7.8–8.0) and 15°C. The present study examined the effects of variation in O2-affinity on the setting of . We present data obtained, at rest and during normoxia, in crustaceans chosen for their distinctly different blood O2-affinities with P50 varying from 0.2 to 2.0 kPa.Seven species of freshwater (fw) and/or seawater (sw) crustaceans were examined from January to October 1991: Procambarus clarkii (fw), Astacus leptodactylus (fw), Eriocheir sinensis (fw and sw; studied in sw), Carcinus maenas (sw), Homarus vulgaris (sw), Cancer pagurus (sw), Maia squinado (sw) and Necora puber (formerly Macropipus puber, sw). All were intermoult animals (see Table 1 for N values). They were either collected locally (C.m., E.s., N.p.) or obtained from commercial suppliers (P.c., A.I., H.v., C.p., M.s.) and adapted to laboratory conditions for at least 1 week prior to experimentation. During the maintenance period they were supplied with aerated sea water or fresh water at a temperature ranging from 8 to 22°C depending on the season. Experiments on P. clarkii and A. leptodactylus were performed in Strasbourg tap water (see Massabuau et al. 1991, for ionic composition). All experiments in sea water were performed in Arcachon sea water (salinity 30–32%o). The experimental conditions were as follow: temperature 15.0±0.5°C; ; and pH7.8–7.9 or 8.3–8.4 depending on the titration alkalinity, which was 1.8-2.0mequivl−1 in sea water and 4.4 in fresh water. At least 5 days before experiments began animals were starved, adapted to the experimental conditions, and equipped for arterial blood sampling. The latter consisted of drilling a hole in the shell above the heart. A thin layer of cuticle was left in place and a piece of rubber was glued over it (Butler et al. 1978). Care was taken not to disturb the animals during this 5-day period. The experiments consisted (in less than 30s) of removing an individual from water without disturbing the other animals (i.e. without inducing escape behavior), puncturing the rubber membrane with a capillary glass tube equipped with a needle and collecting arterial blood (180pl). This sampling technique was critically assessed in Forgue et al. (1992) and considered to provide true in vivo, values comparable to those previously obtained with the use of extracorporeal techniques (Massabuau and Burtin, 1984; Forgue et al. 1989). All sampling was performed between 10:00 h and 18:00 h. Each individual was sampled only once. Values of and arterial blood pH (pHa) were determined within 3 min of sampling using a Radiometer polaro-graphic electrode and a pH Radiometer G299A capillary electrode thermostatted at 15°C. Immediately after arterial blood sampling, 500 μl of venous blood was collected from the base of a walking leg to determine total blood copper concentration, [Cu]b (Boehringer kit no. 124834). This was used as an index of the haemocyanin content. The O2-binding curves of whole blood in P. clarkii and in E. sinensis were determined at 15°C using the diffusion chamber technique described by Sick and Gersonde (1969) and gas-mixing Wösthoff pumps.The frequency distribution of all measured values is presented in Fig. 1. In the resting conditions of the experiments, most of the values are low and the data are not normally distributed. The most frequently measured values (i.e. the modes) are in the range of 1–3 kPa. They are similar in all species examined, irrespective (i) of the fact that some are so-called ‘active’ animals (N. puber, C. maenas) while other are ‘sluggish’ (M. squinado, C. pagurus) and (ii) of variations in sampling period (summer/autumn/winter, including the corresponding temperature changes between holding and experimental tanks). Note that higher , values of up to 12 kPa were also occasionally observed. Corresponding pHa values are presented in Table 1 with mean values for , and other measured variables. Values of P50 from various authors are also reported for similar pH values. These results obtained in crustaceans are completed by data previously obtained in the mollusc Anodonta and the fish Silurus (open bars in Fig. 1; Massabuau et al. 1991; Forgue et al. 1989). Distributions and modes of are similar in animals from the three phyla. As illustrated in Fig. 2A, the modes of are independent of P50 at physiological pH. In Fig. 2B, the position of the modes is plotted on O2 saturation or O2 concentration curves vs for whole blood. Although all species exhibited rather low values of , all values are located in the upper half of the curves. The lowest saturation percentage, in the range 50–80%, was observed in Necora, an animal considered to be very ‘active’ and ‘O2 sensitive’. The highest saturation, 98–99%, was observed in Procambarus, an animal thought to be very O2 resistant and known for its burrowing habits.In the analysis of the present results two points are of particular importance. First, data were obtained in a group of crustaceans and in a teleost fish which display a homogeneous level of resting O2 consumption (Table 1). Second, in the resting (incidentally starved) and normoxic conditions we used, the constraints on the respiratory system were minimal and was allowed to drift spontaneously to a resting value following handling (McMahon, 1985, and personal observation). Both points allowed us specifically to study the effect of the blood O2-affinity on the setting of at rest in water-breathers. We show that, in these conditions, is mainly kept in the range 1–3 kPa, regardless of the blood O2-affinity (for P50 varying from 0.2 to 2.0kPa). This apparent set point of is similar to that already reported in the mussel A. cygnea, which has no respiratory pigment but a seven-to 10-fold lower O2 consumption (Table 1). Arguing from a largely theoretical basis, Malte and Weber (1987) discussed the effect of the shape and position of the oxygen equilibrium curve on extraction and ventilatory requirement in normoxic fishes. They compared the effects of low and high P50 (0.5 and 4 kPa) when O2 saturation in the arterial and venous blood ( and ) were fixed at 95 and 60%, respectively. By comparing theoretical curves with experimental P50 from carp and trout (P50 of 0.6–0.9 and 2.6–2.9 kPa, respectively), they concluded that a high affinity may allow an increase in O2 extraction from water and a reduction in ventilation. In other words, a low is sufficient to saturate a high-affinity pigment and this can be achieved with a lower ventilatory flow rate. Starting with the hypothesis that and are fixed, these conclusions are not in question. But the main difference from our results is that we did not find a fixed , value. On the contrary, it is that remained in a narrow range as an apparent controlled value (Fig. 2A). Any further detailed comparison appears rather speculative because (i) the maximum theoretical P50 of 4 kPa used by Malte and Weber (1987) is quite high in comparison to the presently studied range and (ii) trout have a much higher metabolic level than the animals we studied. However, it is clear from our results that reaching full saturation of the respiratory pigment is not a prerequisite, at least in resting and normoxic conditions. To summarize, based on both present findings and previous results cited at the beginning of this report, all the water-breathers we studied maintained at rest in the range 1–3 kPa (at 13–15°C) irrespective of species and phyla, marine or freshwater origin, season (taking into account the 5-day acclimation period at 15°C), organization of the respiratory system (gill type, ventilatory pump, open versus closed circulatory system), presence or absence of respiratory pigment and, when present, concentration of respiratory pigment and blood O2-affinity for P50 in the range of 0.2–2.0 kPa. Our present experiments were performed in normoxia but this was also observed at various values of in A. leptodactylus, S. glanis and A. cygnea.In conclusion, the frequent occurrence of low values in our experimental conditions, only slightly above the anaerobic threshold (0.7–1.2 kPa) determined in deep hypoxia (Forgue et al. 1992), appears to be a strong intrinsic characteristic of gas-exchange regulation in resting water-breathers. The infrequent occurrence of higher values must, however, also be taken into account in the analysis of the overall gas exchange.The animals we studied represent a wide spectrum of physiologically different water-breathers, but are not representative of all species and all physiological conditions. Comparison with similar data obtained in animals with higher metabolic rates should be very interesting.The crew of the Côte d’Aquitaine (INSU-CNRS) provided the crabs Eriocheir sinensis. Dr John Simmers corrected the English manuscript. J.F. was supported by a grant from Ministère de la Recherche et de la Technologie.