The Regulation of Haemocyanin Oxygen Affinity During Emersion Of the Crayfish Austropotamobius Pallipes: II. An Investigation of In Vivo Changes in Oxygen Affinity

Voluntary emersion behaviour by crustaceans which are subjected to hypoxic conditions has been demonstrated to benefit some species by maintaining a relatively high O₂ content in prebranchial haemolymph, although the often fell (Taylor, Butler & Sherlock, 1973; Wheatly & Taylor, 1979; McMahon & Wilkes, 1983). The freshwater crayfish Austropotamobius pallipes is one species which has been observed to leave hypoxic water and to breathe air (Taylor & Wheatly, 1980). In A. pallipes the initial acidosis of the haemolymph during air exposure was demonstrated to be of mixed respiratory and metabolic origin (Taylor & Wheatly, 1981a) and during prolonged emersion to be compensated by an elevation of buffer base.

The acidification of the haemolymph in A. pallipes was demonstrated (Taylor & Wheatly, 1981a) to reduce, via the Bohr effect, the oxygen affinity of the haemolymph. A discrepancy between in vitro and in vivo estimates of oxygen affinity could not, however, be accounted for. Subsequent to this work Truchot (1980) reported that L-lactate can increase the oxygen affinity of some crustacean haemocyanins and this effect may help to explain some of the discrepancies observed during earlier studies. This effect has now been confirmed for several different species and occurs to a marked extent in the haemolymph of A. pallipes (Morris, Tyler-Jones & Taylor, 1986).

The potentiation of haemocyanin oxygen affinity by Ca²⁺ is well documented and several earlier in vitro studies are referred to in the previous paper (Morris et al. 1986). In that study we demonstrated that in vitro the effects of Ca²⁺ and L-lactate on the oxygen affinity of A. pallipes haemocyanin were not independent. The investigations were carried out using dialysed haemolymph and little could be concluded as to the actual events in vivo. The present study, therefore, considers the changes in both inorganic cation and L-lactate concentrations during aerial exposure and recovery and the concomitant changes in the haemolymph oxygen affinity in A. pallipes. The interactions of the Ca²⁺, L-lactate and H⁺ effects in vivo are reported and discussed in the light of previous findings.

Specimens of the common British crayfish (weight range 11–34 g), Austropota-mobius pallipes (Lereboullet) were collected and maintained, prior to the experiments, at 15 ± 1°C in aquarium tanks with gravel beds supplied with fresh running water (detailed in Taylor & Wheatly, 1980, 1981a). From this group of acclimatized crayfish, 15 intermoult animals were used to investigate the effects of aerial exposure (70% humidity) on the ionic composition and oxygen transporting properties of the haemolymph.

The haemolymph was quickly withdrawn from the pericardial space via the arthrodial membrane at the posterior edge of the carapace of three animals prior to the initiation of 24 h aerial exposure. The experimental design was similar to that of Taylor & Wheatly (1981a) and each animal was used once only. During the experiment haemolymph was taken from three different crayfish, on each occasion, after 2h aerial exposure, after 5h aerial exposure and after 1 h immersed recovery from 24 h emersion. Haemolymph was taken from a further two crayfish after 8h immersed recovery. These times were selected in order to incorporate the maximum and minimum values of haemolymph pH and L-lactate concentration (Taylor & Wheatly, 1981a) that occur during the immersion-emersion immersion series. From each of the samples taken a sub-sample was frozen for subsequent analysis of - ion composition and L-lactate content. The main part of each sample was frozen (–70°C) in separate Eppendorf vials and transported under solid CO2 to Düsseldorf where the effects of aerial exposure on the oxygen transporting properties of the haemolymph were investigated.

The sub-samples of haemolymph taken from individual A. pallipes were analysed first for [Ca²⁺], [Mg²⁺] and [K⁺] using an atomic absorption spectrophotometer (Pye Unicam SP9). The concentrations of these ions were also measured in a preliminary experimental series carried out using crayfish from the same population as those used in the present study and exposed to the same period of aerial exposure as used here. Secondly, the haemolymph was in each case analysed for L-lactate according to the method of Gutmann & Wahlefeld (1974) with attention to the modification made by adding EDTA to the assay mixture (Engel & Jones, 1978; Graham, Mangum, Terwilliger & Terwilliger, 1983). All determinations were made in triplicate. The concentration of haemocyanin was measured in each of the samples, using a spectrophotometer to determine the extinction peak near to 335 nm, and calculated assuming an extinction coefficent of 2·69 (Nickerson & van Holde, 1971).

Oxygen equilibrium curves were constructed for the haemolymph taken from each of the individual crayfish sampled during either immersion or exposure to air. The curves were made using a spectrophotometric method on 8-μ l samples in a diffusion chamber (Sick & Gersonde, 1969) following the methodology described by Bridges, Morris & Grieshaber (1984). Details of this method and its application to the haemolymph of A. pallipes were provided in the previous paper (Morris et al. 1986). All determinations were made at 15°C. The values of P₅₀ and of the cooperativity (n₅₀) were determined by the regression analysis of the saturation values between 25 and 75% according to the Hill equation. Unless otherwise indicated, values are given as means ± S.D.

The concentration of haemocyanin in the haemolymph of the experimental crayfish varied between 51 and 62 mg ml⁻¹ but it was not possible to correlate haemocyanin concentration with the time of sampling. The measured concentrations of K⁺, Mg²⁺ and Ca²⁺ ions in the haemolymph varied as a result of aerial exposure, that of Ca²⁺ extensively (Fig. 1). The mean values for [Ca²⁺], [K⁺] and [Mg²⁺] in the haemolymph of quiescent submerged A. pallipes were 10 · 24 ± 2 · 4mmoll⁻¹, 2 · 81 ± 0 · 29mmol I⁻¹ and 2 · 16 ± 0 · 56mmoll⁻¹, respectively. After 5h aerial exposure, the concentrations of these ions had increased significantly to: Ca²⁺, 13-4 ± l · 5mmoll −¹; K⁺, 4 · 48 ± 0 · 40 mmol I⁻¹ and Mg²⁺, 2 · 43 ± 0 · 56 mmol T¹. The concentration of Ca²⁺ increased to 47 mmol I⁻¹ after 24 h in air but, after 1 h immersed recovery, decreased to 28 · 1 ± 7 · 7 mmol I⁻¹. This was still significantly elevated with respect to the control values. Subsequent measurements showed, however, that after 5 h immersed recovery the concentrations of these ions had returned to near normal levels.

Similar changes in the concentration of L-lactate occurred in the haemolymph of aerially exposed A. pallipes (Fig. 1). In contrast to the relatively small increase in inorganic ion concentration that occurred in the first 5 h in air, the concentration of L-lactate increased dramatically to 8-75 ± 0-45 mmol I⁻¹ compared to a value of only 0·58 ± 0·32 mmol I⁻¹ in the control animals. It is also apparent from Fig. 1 that this increased L-lactate concentration occurred during the period in which the haemolymph exhibits a large acidosis. In the subsequent period between 5 and 24 h air exposure, haemolymph acidosis decreased together with [L-lactate] whereas [Ca²⁺] increased. Subsequent reimmersion resulted, however, in the concentration of L-lactate in the haemolymph rising to 10·35 ± 0·57 mmol I⁻¹, which can be correlated with a period of slight haemolymph alkalosis previously reported by Taylor & Wheatly (1981a,b). After 5 h immersion, the concentration of L-lactate had fallen to 4·04 ± 2·17 mmol I⁻¹ and the other haemolymph variables returned to the values measured for settled, submerged crayfish (Fig. 1).

The dependence of oxygen affinity (logP₅₀) on the pH of the haemolymph from individual crayfish was calculated by least-squares regression analysis (Table 1). In order that any change in oxygen affinity could be clearly observed and to minimize the effects of individual variation, the data for each of the five groups (A-E) were pooled (Fig. 2). It was immediately apparent that relatively large changes in the oxygen affinity of the haemolymph did occur in aerially exposed A. pallipes. Analysis (covariance) of the combined data showed that oxygen affinity varied significantly (P < 0·01) but it was not possible to demonstrate a statistically significant variation in the magnitude of the Bohr factor. Although the observed changes in the magnitude of the Bohr factor (φ) were not large (Fig. 2) there was a noticeable change in the dependence of O₂ affinity on haemolymph pH (Δ φ = -0·25). The changes in the value of φ throughout the immersion-emersion series could not be correlated with any of the other measured haemolymph parameters.

Considering each of the five regression lines, describing the pooled data, at a constant pH (pH 7·9, in vivo value for control animals) it was found that the affinity responded directly to the changing condition of the haemolymph during aerial exposure. After only 1 h in air, the haemolymph oxygen affinity had decreased (ΔlogP₅₀ = 0·06 Torr), possibly in response to the slight decrease in [Ca²⁺] that occurred (Fig. 1), despite the increase in [L-lactate]. During the next 4h of aerial exposure, when both [L-lactate] and [Ca²⁺] increased, the O₂ affinity also increased (AlogP₅₀= -0·22 Torr). This did not represent, however, the maximum haemolymph affinity measured, since when the crayfish were returned to water the affinity of the haemolymph exhibited a further increase (AlogP₅o = -0·09 Torr, pH 7·9) that could be correlated with the maximum concentrations of L-lactate and a high concentration of Ca²⁺. In the same way as haemolymph ion levels returned to near normal during immersed recovery, so did the O₂ affinity of the haemolymph.

The cooperativity (n₅₀) of the haemocyanin in the different haemolymph samples was very similar (Fig. 2). There was no significant dependence of n₅₀ on haemolymph pH, although group D animals provided haemolymph containing high concentrations of both L-lactate and Ca²⁺.

A number of studies have examined inorganic ion concentrations in the haemolymph of air-breathing crabs (e.g. Lutz, 1969; Taylor & Butler, 1978; Burggren & McMahon, 1981; Wernburg & Goldenberg, 1984; Wheatly, Burggren & McMahon, 1984), but few have considered the effects of fluctuation in concentration on O₂ transport. Interestingly, deFur, Wilkes & McMahon (1980) showed that Cancer productos, when exposed to air, exhibited increased [Ca²⁺] in the same way as A. pallipes in the present study. The large difference in the magnitude of the change in [Ca²⁺], compared to the changes exhibited by the other ions, implies that [Ca²⁺] is affected specifically.

There are now several published reports to indicate that sustained tissue and haemolymph acidosis in crustaceans initiates a compensatory increase in circulating HCO₃⁻ ions (Truchot, 1975; McMahon, Butler & Taylor, 1978; Wheatly & Taylor, 1979; deFur et al. 1980; Henry, Kormanik, Smatresk & Cameron, 1981; McMahon & Wilkes, 1983; deFur & McMahon, 1984b); this has also been shown to occur in A. pallipes (Taylor & Wheatly, 1981a). The most likely source for both the Ca²⁺ and HCO3⁻ ions is exoskeletal CaCO₃, since the other possible source of bicarbonate ions — branchial exchange - can play only a minimal role during air breathing (Henry et al. 1981; deFur & McMahon, 1984b).

Changes in the haemolymph concentration of L-lactate were especially interesting in that they were indicative of the metabolic state of the animal. The contribution of anaerobiosis to energy production during aerial exposure of A. pallipes has been discussed at length by Taylor & Wheatly (1981a). The resumption of aerobiosis is reflected by the subsequent decline in L-lactate which is most probably reoxidized to pyruvate. The subsequent peak in [L-lactate] when the crayfish are reimmersed in normoxic water is less explicable, but Taylor & Wheatly (1981a) point out that this may represent a washing out of any L-lactate previously sequestered by the tissues. It is significant that this phenomenon occurs at a time of haemolymph alkalosis caused by a hyperventilatory response to reimmersion, making it appear unlikely that lactic acid is actually produced at this time. The work involved in hyperventilation could lead, however, to some transient functional anaerobiosis.

The importance of anaerobiosis in emersed A. pallipes becomes reduced as oxygen delivery to the tissues becomes restored. Taylor & Wheatly (1981a) indicate the role of water in the branchial chamber in initially reducing O₂ uptake of the exposed crayfish and also stress the importance of changes in the gas transporting properties of the haemolymph. These authors showed that, during 24 h aerial exposure, both and recovered from the almost complete depletion of haemolymph oxygen that occurred during initial exposure. This took place, however, without a concomitant increase in or (Taylor & Wheatly, 1981a,b) and to an extent that cannot be explained by the modest Bohr effect. Morris et al. (1986) postulated that both Ca²⁺ and L-lactate have important roles in regulating haemocyanin oxygen affinity in A. pallipes but that their effects are interdependent. By fitting the haemolymph variables measured in this study to the graphical analysis made by these authors for dialysed haemolymph, it has been possible to predict what the likely changes in haemocyanin oxygen affinity would be during the experimental series (Fig. 3). It is apparent from the model shown in Fig. 3 that the O₂ affinity of dialysed haemolymph would increase during aerial exposure (P₅₀ decreases from 6 to 2 Torr). This is due primarily to an initially high, but subsequently declining, [L-lactate] and to a gradual elevation of [Ca^z+]. This change in affinity, during the initial phases of aerial exposure, is opposite in direction to the change expected when only a Bohr shift occurs. The subsequent alkalosis results in a left shift of the O2 equilibrium curve and thus an increase in oxygen affinity. The predicted increase in oxygen affinity (ΔlogP₅₀, –0·5) is, however, considerably greater than expected from a Bohr shift alone (ΔlogP₅₀, –0·19). A return of the various ion concentrations during immersed recovery to more normal values is expected to return O₂ affinity to near resting values.

The prediction described above is also compared to the actual determined oxygen affinities at the in vivo pH value (Fig. 3) of haemolymph in A. pallipes. First, the oxygen affinities predicted for dialysed blood are less than those determined for the native haemolymph samples. This is due to the effect of unidentified plasma factors (UF) increasing the oxygen affinity. The presence and effect of this factor(s) have been investigated in a number of recent studies (Truchot, 1980; Mangum, 1983a,b,Bridges et al. 1984; Morris, Bridges & Grieshaber, 1985a) and the effect of UF in the haemolymph of A. pallipes has been discussed by Morris et al. (1985b). It can be seen from Fig. 3 that this effect does not remain constant and, although this could be due to a deficiency in the in vitro analysis, possible interactions with other cofactors of haemocyanin oxygen affinity might occur, thus imparting a true modulating role to UF (see also Bouchet & Truchot, 1985).

In general, the predicted trends in oxygen affinity reflect the measurements made in this study confirming the interdependent effects of Ca²⁺ and L-lactate in vivo to be those shown in vitro (Morris et al. 1986). This is supported by deFur & McMahon’s (1984a) conclusion that the effect of L-lactate on C. productus haemocyanin was ‘mitigated’ during emersion, possibly by an increased concentration of Ca²⁺.

It can be concluded that when A. pallipes is exposed to air, several compensatory mechanisms are employed in order to maintain energy production - initially, through anaerobiosis, and secondly, by restoring oxygen delivery to the tissues. In the longer term, A. pallipes appears to adjust to air breathing by compensating the haemolymph acidosis by mobilizing CaCO₃ to increase both the level of circulating HCO₃⁻ and Ca²⁺. Large changes in the O₂ content can be achieved by small changes in affinity because of the already low P₅₀ of the haemocyanin. The restoration of O₂ delivery to the tissues reduces the contribution of anaerobiosis, and an oxygen debt appears to be repaid without the animal having to return to the water. Having adjusted to air breathing, subsequent reimmersion would appear to require readjustments of the gas-transporting properties of the haemolymph, which are reflected in the transient fluctuations in haemolymph parameters. Finally, it is possible that fluctuations in haemolymph ion concentrations play a role in a variety of stress situations in different crustaceans. The evolution mA. pallipes of such a marked sensitivity of the haemocyanin to [Ca²⁺] indicates that this species appears to depend on a high haemocyanin oxygen affinity when and are low.

We would like to thank Ms A. Lundkowski for technical assistance. Financial support for this work was provided by the Royal Society London (SM, European Exchange Fellowship), the Scientific and Engineering Research Council (RT-J, Research Studentship) and the Deutsche Forschungsgemeinschaft (CRB, Grant Gr 456/10–1).