Eco-Physiological Studies of an Intertidal Crustacean, Pollicipes Polymerus (Cirripedia, Lepadomorpha): Aquatic and Aerial Respiration

The Pacific gooseneck barnacle, Pollicipes (Mitella) polymerus Sowerby, inhabits the upper two-thirds of the intertidal zone on highly exposed rocky shores along the west coast of North America (Cornwall, 1925). Pollicipes is a sessile animal attached to hard rocky substrate, but also to shells of Mytilus or the peduncles of other Pollicipes. Being sessile, Pollicipes and other intertidal cirripeds must tolerate gross changes in ambient conditions in order to be successfully adapted. A combined field and laboratory study of Pollicipes has been undertaken in an attempt to evaulate the qualities making this animal successful in its intertidal habitat. In an earlier paper (Fyhn, Petersen & Johansen, 1972) problems of temperature change, desiccation and osmotic stress in Pollicipes have been investigated. The present report deals with aspects of gas exchange in Pollicipes both in water and in air under changing environmental conditions.

In the region of study at Friday Harbor, Washington State, U.S.A., spring and summer have prevailing westerly to north-westerly winds resulting in a dry season from May to mid-August. The wet season, having more south-westerly winds, ranges from October to February, abating in spring. Relative humidity during winter ranges from 90% at night to 75% in the afternoon. Corresponding figures for spring and fall are 85–60%, with summer humidities ranging from 85% to as low as 20% when easterly winds prevail.

Sea-water temperatures in the Strait of Juan de Fuca and northern Puget Sound show only small annual variations, from 7°C in February and March up to 13 and 14 °C in July and August. Air temperatures, however, change considerably throughout the year, from −15 °C in January to 37 °C in July.

The tides at Friday Harbor and vicinity show marked diurnal inequality. In the winter months (January-February) low tides usually occur at night, exposing some animals until 02.00 h and even 03.00 h. During summer (July-August) low tides occur commonly between 07.00 and 11.00 h, exposing part of the Pollicipes population until 17.00 h.

The Pollicipes populations in the Friday Harbor region are thus exposed to considerable variations in ambient conditions, particularly since low tide can coincide with extreme temperatures.

Pollicipes polymerus were collected and maintained according to the procedure described by Fyhn et al. (1972). All animals were obtained between April and July from the populations distributed along the intertidal zone at Eagle Point and Eagle Cove, on the west coast of San Juan Island, State of Washington. Specimens attached to shells of Mytilus were preferred, since these could be isolated for individual study of intact unharmed animals. Animals were kept in the laboratory submerged in aquaria with running unfiltered sea water. The results presented were obtained on animals studied within a week of collection, although Pollicipes appeared healthy for several weeks under laboratory conditions. Animals with the exuvia attached were excluded, but no other consideration was given to effects of the moult cycle. During May the ovaries mature and ovigerous lamellae are present in the capitulum from late May. Haemolymph samples which contained eggs were discarded. The animals used ranged in weight from 10 to 40 g. During the experimental period the temperature of the sea water ranged between 8·5 and 10°C while the salinity varied between 28·5 and 30·5%0.

Samples of haemolymph were collected by percutaneous puncture of the peduncular sinus. Sample sizes of 1–2 ml prevented sampling errors due to poor mixing of the haemolymph. The samples were stored on ice during transport to the laboratory for analysis of oxygen tension⁠, carbon dioxide tension and pH within 1 h of collection. No significant changes in haemolymph gas tensions or pH occurred in samples stored on ice for 2 h.

Gas exchange was measured by closed respirometry using glass respirometers. Five to seven animals of comparable size (15–25 g) were used per respirometer. Experiments were carried out at 2,10, 20 and 26–28°C with air-exposed animals, and at 10 and 20°C with animals submerged in filtered sea water of salinity 28·5–30·5%. The experiments lasted for 6–8 h with air-exposed animals and for 4–5 and 8–10 h with submerged animals at 20 and 10°C, respectively. The respirometers were kept submerged in a thermostatted bath.

Water or air in the respirometers was periodically sampled and analysed for gas tensions and pH. A compensation chamber prevented pressure changes in the respirometers from water sampling. The samples were analysed at room temperature (20 ± 1°C) with a Radiometer PHM 71 acid-base analyser equipped with Radiometer oxygen and carbon dioxide electrodes, and with a Radiometer pH micro-electrode unit. Temperature corrections of oxygen tension readings were made to take account of the temperatures of the animals using solubility coefficients for oxygen in sea water (Harvey, 1966). No temperature corrections were made of the readings of pH and carbon dioxide tensions. Oxygen uptake was calculated using solubility coefficients for oxygen in sea water after Harvey (1966) and computed on a live-weight basis. The Radiometer equipment was also used for analyses of haemolymph gas tensions and pH.

In order to measure the rate of gas exchange across the peduncle cuticle, the upper half to two-thirds of the peduncle were cut off and prepared according to the procedure of Fyhn et al. (1972). The cut off peduncles were filled with either hypoxic or hyperoxic sea water with a 27-gauge needle through the stopper. Care was taken to assure the preparations to be leakproof and that all air bubbles were removed. The preparations were then placed in stirred sea water of either high or low oxygen tension in order to set up a gradient across the peduncle cuticle. After 2 h samples of the internal fluid were withdrawn and analysed for oxygen tension, allowing computation of the rate of gas transfer.

Lactic acid analyses of the haemolymph were made with the Rapid Lactate Stat-Pack (Calbiochem). The analyses were done in duplicate with 100 μl of haemolymph for each measurement.

Oxygen content of haemolymph and sea water was determined according to the method of Tucker (1967).

Haemolymph was sampled from individual animals throughout the tidal cycle in the field and analysed for oxygen tension, carbon dioxide tension, and pH. Each animal was marked and sampled only once. Fig. 1 shows the oxygen tension of the haemolymph for two series of samples, one taken in April and one in June. The oxygen tension increases during air exposure and drops again during the succeeding submersion. The effect of increased temperature during air exposure (Fyhn et al. 1972) is taken into account by correcting all oxygen tensions to 10° C, the sea-water temperature. The increase in oxygen tension is therefore not a result of temperature increase but indicates an efficient gas exchange during air exposure. The same procedure for sampling and analysis was employed on all samples in order to minimize variations due to ambient conditions or to individual animals. The April series and June series are for immature and mature animals, the latter with ovigerous lamellae present and the differences between the two series may be due to this fact. In both cases, however, the haemolymph oxygen tension is significantly higher during air exposure than when submerged.

Carbon dioxide tension and pH of haemolymph sampled during the tidal cycle are shown in Fig. 2. Again the data from the April and June series show the same trend, varying only in magnitude. Air exposure is accompanied by an increase in carbon dioxide tension and a concomitant decrease in pH. During resubmersion the haemolymph carbon dioxide tension and pH are again restored to values typical of the submerged condition. In the June series the carbon dioxide tension more than doubles during the 9 h of air exposure and the mean pH drops from 7·49 to 7·23, signalling a considerable acidification of the haemolymph.

Analyses of lactic acid in the haemolymph showed no significant variation during the tidal cycle (Table 1).

The oxygen uptake of Pollicipes in relation to changing ambient conditions were studied in the laboratory. Fig. 3 shows the oxygen uptake in relation to temperature for air-exposed animals and for animals submerged in normoxic sea water (= 150 mmHg). Oxygen uptake increases with temperature both for air-exposed and submerged animals. Air-exposed animals have a much higher respiratory rate than submerged animals at each tested temperature, amounting to five times higher uptake at 10° C and three times at 20° C. The temperature effect on the oxygen uptake rate thus is different in submerged and in air-exposed animals. While submerged animals have a Q₁₀ = 2·5 in the temperature interval 10–20 °C, air-exposed animals have Q₁₀ = 1·6 in the same temperature interval. The oxygen uptake of air-exposed animals shows a decreased dependency with increasing temperature: a Q₁₀ of 2·2 in the interval 2–10 °C is reduced to a Q₁₀ of 1·6 between 10 and 20° C and 1·3 between 20 and 27° C.

The effect of hypoxic and hyperoxic water on the oxygen uptake of Pollicipes at 10 and 20° C is shown in Fig. 4. At both temperatures Pollicipes shows oxygen conformity, i.e. a reduced uptake as the oxygen tension declines.

A possible presence of an oxygen debt after 3–4 h exposure to hypoxic sea water (⁠ = 40 mmHg) was tested at 10 and 20° C. No compensatory increase in oxygen uptake was discernible when the animals were transferred to normoxic water. At 10° C the oxygen uptake (mean ± S.E.) was 2·5 ± 0·5 μl O₂/g/h before hypoxic exposure and 2·4+ 0·1 after, and at 20° C the corresponding values were 4·4 ± 1·2 and 4·2 +0·3 μl O₂/g/h.

The effect of ambient oxygen tension on haemolymph oxygen tension, carbon dioxide tension and pH was studied in laboratory experiments at 10° C on animals collected in June. Control animals were used to check survival and normal behaviour following experimental exposure. Animals were selected from those collected on the same or the previous day, preference being given to fairly large animals (25–30 g), so that large haemolymph samples could be obtained. The samples were analysed immediately after collection. Two different approaches were used. One consisted of placing the animals in normoxic sea water and then bubbling oxygen or nitrogen through the water. In other experiments the animals were transferred from normoxic water directly to either hypoxic or hyperoxic water. The latter approach allows a better evaluation of the total time the animals are exposed to specified conditions. The duration of exposure to either hypoxic or hyperoxic conditions ranged from to 4 h with a mean of 3 h. The results obtained following the two approaches were overlapping and a composite picture of all results is presented in Fig. 5. In the normoxic state (⁠ = 150 mmHg) the haemolymph gas tensions and pH have values similar to those found for submerged animals in the field (Figs. 1, 2). Hypoxia (⁠ = 25 mmHg) correlates with a decreased oxygen tension of about 7 mmHg while haemolymph carbon dioxide tension and pH retain their normoxic values. Hyperoxia = 325 mmHg) results in a small increase in haemolymph oxygen tension to a mean of 36 mmHg, a pronounced increase in haemolymph carbon tension and a concomitant decrease in haemolymph pH. This finding suggests that the metabolic rate increases during hyperoxia, as implied by the oxygen uptake (Fig. 4). The oxygen conformity behaviour may also partly be responsible for the relatively modest increase in haemolymph oxygen tension during hyperoxia. No deleterious effects on overall behaviour were apparent following exposure to either hypoxic or hyperoxic conditions.

Oxygen content analyses showed that Pollicipes haemolymph equilibrated with air at room temperature had an equal oxygen capacity to isosmotic sea water, showing that there is no functional respiratory pigment in the haemolymph.

Four animals were used for measurement of the permeability of the peduncle cuticle to oxygen. Two prepared peduncles were filled with normoxic sea water and placed in hypoxic water for 2 h whereafter external and internal oxygen tensions were determined. Two other preparations were filled with hypoxic water and placed in normoxic water for 2 h followed by oxygen tension measurements. The sea-water salinity was 30% and temperature 10 ° C. The results (Table 2) show that the cuticle in itself does not constitute a diffusion barrier to oxygen.

An intertidal animal like Pollicipes must be able to exchange gases alternately with water and with air during the tidal cycle. In general, respiratory organs are adapted to serve efficient gas exchange in one of these media and will perform less well with the other, compelling the animal to accumulate an oxygen debt or to depend temporarily on anaerobic energy production (Mangum & Winkle, 1973). Thus marine invertebrates Exposed to air commonly have a reduced oxygen uptake in comparison to the submerged condition (Grainer & Newell, 1965; Wallace, 1972; Newell, Ahsanullah & Pye, 1972; Petersen & Johansen, 1973). This decreased oxygen uptake mainly arises from the inability of gills and their ventilatory mechanisms to function in air. Often this is correlated with a depression of the metabolic rate during air exposure and a consequent reduction in activity level or other energy requiring behaviour (Newell, 1970, 1973). The present finding of a high respiratory rate in air exposed Pollicipes compared to submerged animals is a notable exception to this general picture and suggests that many intertidal invertebrates may have evolved towards intermittent terrestrialism.

Earlier studies have demonstrated that intertidal balanomorph cirripeds utilize atmospheric oxygen for respiration during air exposure (Barnes & Barnes, 1957; Barnes, Finlayson & Piatigorsky, 1963; Grainer & Newell, 1965). However, the oxygen uptake rate of these barnacles when exposed is much lower than when submerged (Grainer & Newell, 1965). Furthermore, when extensive desiccation occurs these barnacles close their opercular valves and seem to rely on energy derived from anaerobic metabolism (Barnes & Barnes, 1964; Hammen, 1972). The high rate of evaporative water loss from the peduncle of Pollicipes (Fyhn et al. 1972) excludes the possibility of eliminating water loss by closing the opercular valves. However, the large haemolymph volume in the peduncular sinus has been shown to constitute a water reservoir which allows the evaporative water loss to proceed during the intertidal air exposure without reaching lethal limits of desiccation (Fyhn et al. 1972). The increasing oxygen tension of Pollicipes haemolymph during air exposure in the field (Fig. 1) indicates that oxygen uptake continues at a high rate in spite of desiccation. Continuous recordings of heart rate in Pollicipes during air exposure did not reveal any bradycardia (Fyhn, Petersen & Johansen, 1973), which would have been expected if metabolism had been suppressed. The water loss of exposed Pollicipes is effectively replaced during the succeeding submersion (Fyhn et al. 1972) and thus Pollicipes seems well adapted to tolerate the stress of desiccation combined with gas exchange in air.

The oxygen uptake in some species of intertidal gastropods is higher for air exposed animals than for submerged animals by a factor of 1·3–4·4 at temperatures from 18 to 30° C (Micallef & Bannister, 1967; Sandison, 1967). The effect of desiccation on the aerial respiration was not specifically investigated in these studies. However, the finding that the oxygen uptake of the gastropod Monodonta turbinata after 24 h of immersion was higher than after 24 h of air exposure at the same temperature (Micallef & Bannister, 1967) suggests that the aerial respiration is dependent upon water balance in this species. Very few investigations have addressed the effect of desiccation and water balance on the aerial respiration of intertidal animals.

A morphological description of the efficient respiratory surfaces in Pollicipes is not available nor are they well defined for Cirripedia in general. In balanomorph cirripeds with their calcareous shell the gas exchange must be carried out across surfaces within the mantle cavity. The so-called branchiae, the cirri, and the general body surface with its superficial haemolymph sinuses, are all likely sites for respiratory gas exchange (Gutman, 1960; Crisp & Southward, 1961). In PollicipesBurnett (1972) showed that the inner surface of the mantle and the dorsal part of the body with its finger-like appendages, have a plexus of haemolymph sinuses just beneath a thin cuticle and he suggested that these might be involved in gas exchange. Our observations indicate that an appreciable part of the gas exchange may additionally take place across the peduncular wall. Efficient haemolymph circulation would be necessary for the peduncle to be important in overall gas exchange. Fyhn et al. (1973) have recently provided evidence for a high-pressure circulation in Pollicipes. The importance of studying gas exchange in cirripeds with an intact internal circulation is emphasized by this finding. The use of excised bodies in experiments attempting to reveal physiological or biochemical responses relevant to the organismic level (Barnes & Barnes, 1959, 1963, 1964, 1969; Barnes, Barnes & Finlayson, 1963) may give misleading results, even if it is found that the excised bodies survive for many hours (Barnes & Barnes, 1959; Barnes et al. 1963).

During the intertidal cycle the body temperature of Pollicipes may vary extensively from about 10 °C when submerged to 30 °C or more when air exposed on a sunny day (Fyhn et al. 1972). Even at equal temperatures the oxygen uptake of Pollicipes is higher for air exposed animals than for submerged animals (Fig. 3). During a tidal cycle therefore the oxygen uptake of Pollicipes may follow a skewed curve. From a low level when submerged, the oxygen uptake would rise steeply as the animal becomes air exposed and dependent on aerial respiration. An increase in body temperature during period of exposure to air would bring about a further increase. As the incoming tide immersed the animal the respiratory rate would drop sharply due to reversion to aquatic respiration and cooling of the body to 10 °C. Pollicipes thus experiences its largest fluctuations in body temperature during air exposure. On this basis it seems a significant adaptation that the rate of oxygen uptake is much less sensitive to temperature (low Q₁₀ value) during air exposure than in the submerged state. A similar trend has been reported for the intertidal amphineuran Cryptochiton stelleri (Petersen & Johansen, 1973). The mechanism of the stimulatory effect of air exposure on oxygen uptake is unknown but it may be related to accumulation of carbon dioxide in the haemolymph (Fig. 2), since carbon dioxide has been shown to stimulate respiration in other crustaceans (Wolvekamp & Waterman, 1960). In the sessile Pollicipes only slow and irregular body movements are found (Barnes & Reese, 1960) and no increase in activity level is obvious with air exposure. The heart rate does not show any correlation with submergence or air exposure (Fyhn et al. 1973) suggesting that the stimulatory effect of air exposure on oxygen uptake is at the cellular level.

The increase in carbon dioxide tension of the haemolymph of Pollicipes during air exposure (Fig. 2) is in agreement with previous findings in other air exposed aquatic animals (Crustaceans: Howell et al. 1973; Teleosts: Johansen, 1966; Dipnoids: Lenfant & Johansen, 1968) and thus lends further support to the hypothesis of retention of carbon dioxide in the transition from water breathing to air breathing (Howell, 1970; Howell et al. 1973). In Pollicipes the increase in oxygen uptake during air exposure (Fig. 3) will result in an increased carbon dioxide production during intertidal air exposure, and thus increase the carbon dioxide tension of the haemolymph. The increased amount of carbon dioxide could displace the acid-base balance and decrease the pH of the haemolymph (Fig. 2). During resubmersion the relatively high solubility of carbon dioxide in sea water would allow the accumulated carbon dioxide to be washed out, thereby restoring the carbon dioxide tension and pH typical of submerged animals. However, it is also possible that the increased level of oxygen uptake during air exposure could produce an increased amount of organic acids which primarily would decrease the pH of the haemolymph, thereby lowering the solubility of carbon dioxide and resulting in an increased carbon dioxide tension of the haemolymph. No lactic acid accumulation is found in Pollicipes during the tidal cycle (Table 1), and neither is there any increase in amino acid concentration of the haemolymph (Fyhn et al. 1972). However, lactic acid does not seem to be the main end-product of glycolysis in invertebrates (Hammen, 1969, 1972) and other organic acids, especially succinic acid, may perhaps be responsible for the drop in haemolymph pH during air exposure in Pollicipes.

This study was supported by grants GB-1766 from the National Science Foundation and HE-12174 from the National Institutes of Health.

H.J.F. was supported by grant D-6045.8 from the Norwegian Research Council for Science and the Humanities and by Professor S. A. Sexes Legat.