Journal of Experimental Marine Biology and Ecology L 244 (2000) 265±283 www.elsevier.nl/locate/jembe

A comparative study of the oxygen transporting properties of the haemocyanin of ®ve of thalassinidean mud-shrimps

A.C. Taylora,* , C.M. Astall a , R.J.A. Atkinson b aInstitute of Biomedical and Life Sciences, Graham, Kerr Building, University of Glasgow, Glasgow G12 8QQ, UK bUniversity Marine Biological Station, Millport, Isle of Cumbrae KA28 0EG, UK Received 18 May 1999; received in revised form 3 September 1999; accepted 22 September 1999

Abstract

Comparative studies of the haemocyanin of ®ve species of thalassinidean mud-shrimps showed that all ®ve species exhibited a high oxygen af®nity (P50 range 5 1.0±9.3 Torr, at in vivo pH, 108C). Each of the mud-shrimps exhibited a moderately large Bohr coef®cient (21.06 to 2 1.48) and values for the co-operativity of the haemocyanin did not differ greatly between the species

(n50 range 5 2.3±3.8). The highest oxygen af®nities were recorded for the haemocyanin of Callianassa subterranea, Jaxea nocturna and Calocaris macandreae, whereas those for the two species of were slightly lower but were still higher than in many other decapods. The higher oxygen af®nity of the haemocyanin of the deposit feeding shrimps C. subterranea, J. nocturna and C. macandreae compared with that of the haemocyanin of the ®lter feeding upogebiids may be correlated with the fact that conditions within the burrows of the deposit feeders may be more severely hypoxic. The oxygen af®nity of all ®ve species showed a moderate temperate sensitivity (DH 2 56.8 to 2 82.1 kJ mol21 over temperature range 5±108C). Studies of the haemocyanin of one species (C. macandreae) showed that L-lactate did not affect the oxygen af®nity of the haemocyanin as has been reported for a number of other decapod species. The oxygen carrying capacity of the haemocyanin (CHCYO 2 ) was similar in four of the species studied (0.22±0.42 mmol l21 ) but that of C. subterranea was signi®cantly greater (0.83 mmol l21 ). The higher protein concentration of the haemolymph of this species also resulted in the haemolymph having a greater buffering capacity (27.67 mmol l21 pH unit21 ). SDS±PAGE studies of the haemocyanin demonstrated the presence of 3 subunits in the upogebiids but additional subunits were observed in the other species (MW range 5 70 000±90 000 Da). Studies of the association state of the haemocyanin of three of the species showed that the haemocyanin of both C.

*Corresponding author. Tel.: 144-141-330-5972; fax: 144-141-330-5971. E-mail address: [email protected] (A.C. Taylor)

0022-0981/00/$ ± see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-0981(99)00141-0 266 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 macandreae and J. nocturna was present mainly in the form of eikositetramers (24-subunit aggregation state) with some hexameric haemocyanin also occurring. The haemocyanin of Upogebia deltaura, however, occurred as a mixture or eikositetramers and dodecamers.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Callianassa; Calocaris; Haemocyanin; Jaxea; Mud-shrimps; Oxygen transport; Respiratory physiology; Upogebia

1. Introduction

Thalassinidean mud-shrimps form an important component of the macrofauna of sublittoral and some intertidal soft sediments where their burrowing life style makes a major contribution to the bioturbation of these sediments and to nutrient exchange (Pemberton and Buckley, 1976; Koike and Mukai, 1983; Nickell et al., 1996). The burrowing lifestyle adopted by these shrimps offers some protection against predation but exposes them to conditions within the burrow that may be very different from those on the sediment surface (Atkinson and Taylor, 1988; Astall et al., 1997). In particular, the water within the burrows may often be severely hypoxic and may contain signi®cant concentrations of sulphide (Atkinson and Taylor, 1988, Johns et al., 1997). The physiological and behavioural adaptations shown by mud-shrimps have been reviewed by Atkinson and Taylor (1988) but much less is known about the oxygen transporting properties of their haemocyanin. Although Miller and Van Holde and their co-workers have published several detailed studies of thalassinidean haemocyanins, these studies have been restricted to only three species that occur mainly in intertidal locations (Van Holde et al., 1977; Miller et al., 1977; Arisaka and Van Holde, 1979; Miller and Van Holde, 1981a,b). These studies showed that the oxygen af®nities of the haemocyanins of species such as Neotrypaea (5 Callianassa) californiensis, N. gigas and were higher than those of many other non-burrowing decapod (Mangum, 1983a). One particularly interesting ®nding of the research by Miller and Van Holde and their co-workers was the fact that, although the haemocyanin molecule in many decapods exists as either hexamers or dodecamers, the haemocyanin of the thalassinideans studied exists mainly as eikositetramers (24-mers). As part of a larger study of the physiological ecology of mud-shrimps from sublittoral locations, this paper presents the results of a comparative study of ®ve species of mud-shrimps that occur in the same geographical location. These include three primarily deposit feeding species Calocaris macandreae (Bell), Jaxea nocturna (Nardo) and Callianassa subterranea (Montagu) as well as two suspension feeding upogebiids, Upogebia deltaura (Leach) and U. stellata (Montagu) (Nickell and Atkinson, 1995; Nickell et al., 1995; Lindahl and Baden, 1997; Pinn et al., 1998). The aim of this study was to gather further comparative data on the structure and oxygen transporting properties of the haemocyanin of these species thus enabling comparisons to be made with the earlier work on intertidal species. A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 267

2. Materials and methods

2.1. Collection and maintenance of mud shrimps

Upogebia deltaura were collected with an anchor dredge at depths of 6±7 m from Plymouth Sound, Devon, England (508209N, 48139W) and from the Irish Sea (54879N, 38279W). Upogebia stellata were obtained at depths of 20±30 m with an anchor dredge from the north end of Isle of Cumbrae and Calocaris macandreae with an Agassiz trawl, from the Main Channel (between Cumbrae and Bute), Firth of Clyde, Scotland (55899N, 58119W). Specimens of Jaxea nocturna and Callianassa subterranea were obtained from Loch Sween, Scotland (56829N, 58369W) by anchor dredging or by SCUBA diving. All specimens were transported back to the University of Glasgow where they were kept in a re-circulating sea water aquarium (S,32½; T,108C). Individual mud shrimps were placed into small containers (1±2 l) containing suf®cient mud from the sample sites to allow them to construct burrows. Using this method very low mortality rates were recorded.

2.2. Collection of haemolymph

Haemolymph was extracted from quiescent shrimps using a 100 ml syringe (Hamil- ton), the needle (22 g) of which was inserted either into the arthrodial membrane at the base of the walking legs, or beneath the cephalothorax and into the pericardium. Haemolymph samples (10±50 ml) were then transferred to 0.5 ml Eppendorf tubes and kept on ice. As a result of the small volume of haemolymph obtained from each individual (10±50 ml) and the irregularity of specimen capture as well as the paucity of individuals, it was necessary to pool the haemolymph samples to obtain suf®cient haemolymph for some of the analyses. Pooled samples were mixed thoroughly, centrifuged at 13 000 g for 10 min to remove cells and particulate material and kept at 48C or immediately frozen (2208C). Storage of haemolymph at 2 208C has been shown to be preferable to storage at 2 808C, at which temperature the co-operativity of the haemocyanin may be affected in some crustaceans (Morris, 1988; Taylor unpubl. obs.).

2.3. Haemolymph ionic composition

Following dilution with deionised water, the concentrations of Na11 , K , Mg21 and Ca21 in pooled, frozen haemolymph samples from each species were determined using an Atomic Absorption Spectrophotometer (PU 9820, Philips). Lanthanum chloride was added (1:5 v/v) prior to the determination of Ca21 concentrations. The total haemolymph Cl2 concentration was determined by electrochemical titration using a chloride meter (PCLM3, Jenway). These data were used to prepare a physiological saline solution for each species. Since lactate has been shown to be a modulator of oxygen af®nity (Truchot, 1980; Bridges and Morris, 1986), the concentration of L-lactate in pooled haemolymph 268 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 samples was determined using the method of Gutmann and Wahlefeld (1974) with the modi®cations of Engel and Jones (1978).

2.4. Construction of oxygen dissociation curves

In vitro oxygen dissociation curves were constructed on pooled whole haemolymph samples (3 m1) using the spectrophotometric diffusion chamber technique (Sick and Gersonde, 1969). Full details of the procedure used are given in Zainal et al. (1992). Oxygen dissociation curves were constructed for the haemolymph of each species at 5,

10, 15 and 208C by increasing the PO2 of the gas mixture in a stepwise manner while maintaining a constant PCO2 for each curve. Separate samples of haemolymph (100 m1) were simultaneously equilibrated, in a BMS2 (Radiometer) at 108C, against the same gas mixture as supplied to the diffusion chamber. The pH of the haemolymph sample was determined at half saturation (P50) using the microcapillary pH electrode of the BMS 2 connected to a pH meter (215 lon Analyser, Corning). The P50 value and co-operativity (n50) of the haemolymph was estimated using regression analysis of the oxygen saturation values (between 25 and 75%) calculated using the Hill equation. Where possible, the pH of fresh haemolymph samples collected anaerobically from individual shrimps was also determined at 108C using the above method.

2.5. The effect of temperature and L-lactate on haemocyanin oxygen af®nity

The effect of temperature on the oxygen af®nity of the haemocyanin of each species of mud-shrimp was investigated by constructing oxygen dissociation curves at 5, 10 and 158C using the procedure described above. The effect of L-lactate on the oxygen af®nity of the haemocyanin of C. macandreae was investigated by dialysing the haemolymph for12hat48C against differing concentrations of sodium lactate made up in the appropriate physiological saline. Following dialysis, oxygen dissociation curves were constructed at 108C.

2.6. Oxygen carrying capacity

The total oxygen content of the haemolymph (CTOTO 2 ) was measured using haemolymph samples from individual mud-shrimps. The oxygen content of duplicate 10 ml haemolymph samples, equilibrated in the BMS2 against air (to ensure full saturation) at 108C, was determined using the method of Tucker (1967) as modi®ed by Bridges et al. (1979). The oxygen carrying capacity of the haemocyanin (CHCYO 2 ) was calculated by subtracting the physically dissolved fraction of oxygen in the haemolymph from the measured total oxygen content, using an oxygen solubility coef®cient of 0.002 mmol l21 Torr21 (Altman and Dittmer, 1971).

2.7. Haemocyanin sub-unit composition

The subunit composition of the haemocyanin of each species was determined using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). Pooled, A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 269 previously-frozen haemolymph samples were diluted to a ®nal concentration of 1.0, 0.5 or 0.1% with SDS sample buffer (containing 2% SDS, 59% 2-mercaptoethanol, 10% glycerol, 0.0625 M Tris±HCl (pH 6.8) and 0.002% bromophenol blue as reference front) and heated to 1008C for 2±3 min. Aliquots of 30 m1 were loaded onto a 2.5% (w/v N,N9-methylenebisacrylamide) stacking gel and resolved in a 7.5% gel. Electro- phoresis was conducted at a constant current of 15 mA per gel (BioRad mini-gel System). After electrophoresis, the gels were stained for 1±2 h with 0.25% Coomassie Brilliant Blue R and then destained in 7% glacial acetic acid. The molecular weights of the haemocyanin subunits were determined by comparing their electrophoretic mobility with known protein markers. Each gel was calibrated with SDS molecular weight markers in the weight range 29 000±205 000 (MW-SDS-200, Sigma Chemical Co.). A scanning densitometer (GS 300, Hoefer Scienti®c Instruments, San Francisco) was used to determine the number of bands and their relative mobility (Rm). The Rm values were plotted against the known molecular weights and the molecular weight of the unknown protein band was estimated from the calibration curve.

2.8. Association state of the haemocyanins

The association states of the haemocyanins were assessed using fast protein liquid chromatography (FPLC). Unfortunately, no analyses of the haemocyanins of C. subterranea and U. stellata could be carried out due to the limited number of available. A 100 ml aliquot of fresh, not frozen, haemolymph from individuals or from pooled samples, previously diluted with the appropriate physiological saline, was applied to a Superose 6 gel ®ltration column (HR10/30, Pharmacia). The column was eluted with the Ringer's solution (previously ®ltered through a 0.22 mm ®lter) at a rate of 0.2 ml min21 . The column was calibrated with proteins of known molecular weight; blue dextran (MW52 000 000 Da), thyroglobulin (669 000 Da), apoferritin (443 000 Da), alcohol dehydrogenase (150 000 Da) and albumin (66 000 Da) (Sigma Chemical Co.). The activity of naturally occurring protease enzymes in the haemolymph may lead to degradation of the haemocyanin aggregates (Mangum et al., 1987; Terwilliger et al., 1979). Therefore, the association state of the haemocyanin was also assessed for haemolymph treated with a serine protease inhibitor (phenylmethansulphonyl ¯uoride (PMSF) at 1 mmol l21 ®nal concentration) and a trypsin-like serine and cysteine protease inhibitor (leupeptin at 100 mmol l21 ®nal concentration).

3. Results

3.1. Haemolymph ionic composition

The concentrations of the major ions present in the pooled haemolymph samples of ®ve species of thalassinidean mud-shrimps are given in Table 1. The concentrations of these ions did not differ markedly between species although the concentrations of Na1 and Cl2 ions in the haemolymph of Calocaris macandreae were higher than in the other species studied. All of the species examined had high concentrations of magnesium ions 270 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283

Table 1 The concentrations of major ions in the haemolymph of the ®ve species of thalassinidean mud-shrimps studied. All measurements were made from pooled, frozen haemolymph samples Species Concentration (mmol l21 ) Na121 Cl K Mg21 Ca21 Ubogebia stellata 401 457 17.1 40.2 7.8 Ubogebia deltaura 394 446 14.8 43.8 10.7 Jaxea nocturna 358 440 19.8 34.4 6.7 Callianassa subterranea 348 407 22.5 39.9 8.1 Calocaris macandreae 498 520 12.9 45.4 11.6

(34.4±45.4 mmol 121 ). The concentration of L-lactate in pooled haemolymph taken from quiescent individuals was low (0.17±0.58 mmol 121 ) for all ®ve species of mud-shrimp (Table 2). Due to the small size and limited availability of some of the mud-shrimps, data for the in vivo pH of the haemolymph are limited. The available data indicate, however, that the pH of the haemolymph varied slightly between species (Table 2).

3.2. Oxygen carrying capacity

Values for the oxygen carrying capacity of the haemocyanin (CHCYO 2 ) of each of the ®ve species are given in Table 2. C. subterranea had a signi®cantly higher ( p,0.05)

CHCYO 2 than the other mud-shrimps examined. There was no signi®cant difference ( p.0.05) in CHCYO 2 between U. stellata, J. nocturna and C. macandreae although the values for U. deltaura were signi®cantly higher ( p,0.05) than in these species.

3.3. Haemocyanin oxygen af®nity

The relationships between pH and the log P50and between pH and n 50 of the haemocyanin for each of the ®ve species are shown in Fig. 1 and Table 3. Each of the

Table 2

The oxygen-carrying of the haemocyanin (CHCYO 2 ) measured at 108C for the ®ve species of thalassinidean mud-shrimps. The pH of the haemolymph and the concentrations of L-lactate in the haemolymph of each species are also shown. The buffering capacity for the haemolymph (D[HCO3 ]/DpH) calculated from the relationship between PCO2 and pH have been included (for further details see text). All values (except those for buffering capacity) are reported as means6S.D. The number of individual observations are shown in parentheses

Species CHCYO 2 pH L-Lactate Buffer Cap. (mmol l21 ) (mmol l21 ) (mmol l21 pH unit21 ) Upogebia deltaura 0.4260.04 (7) 7.8360.04 (7) 0.4160.03 (2) 25.91 Upogebia stellata 0.2260.01 (2) 7.7360.09 (2) 0.1760.05 (2) 24.23 Jaxea nocturna 0.2760.03 (5) 7.6860.08 (2) 0.3360.01 (2) 24.52 Callianassa subterranea 0.8360.05 (5) 7.7860.06 (5) 0.5860.09 (2) 27.67 Calocaris macandreae 0.2460.05 (25) 7.8160.02 (25) 0.2160.05 (4) 24.11 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 271

Fig. 1. The relationship between log P50and pH (A) and between n 50 and pH (B) at 108C for the haemocyanin of the ®ve species of mud shrimp studied. The equations of the regression lines ®tted to the data are given in Table 3. Upogebia deltaura (h), Upogebia stellata (j), Jaxea nocturna (m), Callianassa subterranea (s) and Calocaris macandreae (d).

Table 3

Regression coef®cients for the relationships between log P50 and pH (at 108C) for the haemolymph of the ®ve species of thalassinidean mud-shrimps presented in Fig. 3. Regression equations are given in the form of log 2 P505a1b(pH), the coef®cient of determination (r ) is also given. All relationships were signi®cant at P,0.05. The Bohr coef®cient (f) is quanti®ed by the b value. Values for calculated the oxygen af®nity of the haemocyanin (P50) at the in vivo pH (Table 2) are also given 2 Species ab r P50 (Torr) Upogebia deltaura 9.09 21.06 0.980 6.2 Upogebia stellata 11.56 21.37 0.941 9.3 Jaxea nocturna 11.57 21.48 0.980 1.6 Callianassa subterranea 10.02 21.29 0.960 1.0 Calocaris macandreae 8.76 21.10 0.941 1.5 272 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283

Table 4

Regression coef®cients for the relationships between log P50 and pH (at 108C) determined from oxygen dissociation curves constructed for haemolymph of Calocaris macandreae following dialysis against differing concentrations of sodium lactate. Regression equations are given in the form of log P505a1b(pH), the coef®cient of determination (r 2) is also given. For each of the regression equations n58 [L-Lactate] ab r2 (mmol l21 ) 2 8.60 21.11 0.941 5 9.29 21.18 0.932 10 7.95 21.01 0.917 15 8.72 21.21 0.962 mud-shrimps exhibited a reasonably large Bohr coef®cient (Table 3). Comparison of the slopes of the regression lines showed, however, that although there were some differences in the Bohr values between species, these were not signi®cant (ANCOVA, P.0.05). The oxygen af®nity of the haemocyanin from all species was quite high although there were some differences between species. In particular, the oxygen af®nities of the haemocyanin of C. subterranea, J. nocturna and C. macandreae were signi®cantly higher than those of the upogebiids at 108C (ANCOVA, P,0.05) (Table 3). There were only small differences in the co-operativity of the haemocyanin among the

®ve species studied (n50 range52.3±3.8). For all species, pH had no signi®cant effect on the value of n50 (Fig. 3). The effect of L-lactate on the oxygen af®nity of the haemocyanin of C. macandreae was examined by constructing oxygen dissociation curves following dialysis of the haemolymph against differing concentrations of lactate. The regression equations describing the relationships between log P50 and pH for the haemolymph of C. macandreae are presented in Table 4. There were no signi®cant differences in either the slopes or the intercepts of the regression lines (ANCOVA, P.0.05) indicating that the presence of lactate ions within this physiological range had no discernible effect of the oxygen af®nity of the haemocyanin. Values for the heat of oxygenation (DH) calculated for the haemocyanins of the ®ve species of mud-shrimp are given in Table 5. The values of DH were approximately

Table 5 Values for the heat of oxygenation (DH) calculated for the haemocyanins of the ®ve species of mud-shrimps studied over the temperature ranges 5±108C and 10±158C. The P50 values used in the calculations were derived from the regression equations ®tted to the relationships between pH and log P50 for each species at the appropriate temperature and in vivo pH Species DH (kJ mol21 ) DH (kJ mol21 ) 5±108C10±158C Upogebia deltaura 265.4 256.2 Upogebia stellata 282.1 263.5 Jaxea nocturna 279.6 266.1 Callianassa subterranea 278.2 253.1 Calocaris macandreae 256.8 243.6 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 273 similar between the species. It was noticeable, however, that the DH values were lower over the temperature range 10±158C than between 5 and 108C.

3.4. Haemolymph buffering capacity

The PCO2 of the haemolymph could not be measured by Astrup titration because of the small amounts of haemolymph obtained from each shrimp. However, an indication of the buffering capacity can be gained by examining the change in pH with PCO2 (at 108C) determined previously during the construction of oxygen equilibrium curves. 2 These data were used to calculate the buffering capacity of the haemolymph (D[HCO3 ]/ 2 DpH). Values for [HCO3 ] were calculated using the Henderson±Hasselbalch equation [HCO32 ] pH 5 pK1 1 log ]]]] aco22? PCO with values for pK12and aco being obtained from the nomograms of Truchot (1976) at the appropriate temperature and salinity. Regression equations describing the relation- 2 ships between [HCO3 ] and pH were then calculated for each of the ®ve species and the values for the buffering capacity of the haemolymph obtained from the slopes of the regression equations. Since the values for the constants pK12and aco were not determined for each of the species, the calculated values for the buffering capacity must therefore be treated with some caution. The data indicate, however, that the buffering capacity of the haemolymph was generally low. The slightly higher buffering capacity of the haemolymph of Callianassa subterranea may be due to the greater haemocyanin content of the blood as indicated by the higher oxygen carrying capacity of the haemocyanin (Table 2).

3.5. Haemocyanin sub-unit composition

Scanning densitometer traces of thalassinidean haemocyanin run under denaturing conditions using SDS±PAGE revealed between three and ®ve sub-units (Fig. 2). The scans for the two upogebiid shrimps showed the presence of three distinguishable sub-units, the molecular weights of which were similar for both species (Table 6). The haemocyanin of Calocaris macandreae and Callianassa subterranea were characterized by four sub-units, whereas ®ve sub-units were present in the haemocyanin from Jaxea nocturna (Fig. 2). The molecular weights for each of the numbered sub-units range from 70 700 to 93 700 Da, and are summarized in Table 6.

3.6. Association state of the haemocyanins

The association states of pooled, fresh haemocyanin were measured for the mud- shrimps Calocaris macandreae, Jaxea nocturna and Upogebia deltaura using fast- protein liquid chromatography (Fig. 3). The haemocyanin of both C. macandreae and J. nocturna was present mainly in the form of eikositetramers (24-subunit aggregation state) (Mr 5approx. 1 900 000 Da) with some hexameric haemocyanin also present 274 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283

Fig. 2. Scanning densitometer traces of the haemocyanin run under denaturing conditions (see text for further details). A5Upogebia deltaura, B5U. stellata,C5Calocaris macandreae, D5Callianassa subterranea,

E5Jaxea nocturna. The individual traces have been aligned against the same Rm scale. A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 275

Table 6 The number of subunits of the haemocyanin, as determined by SDS-PAGE, for the ®ve species of mud-shrimps. The relative mobility (Rm) was determined for each band (Fig. 2) and the molecular weight calculated from a calibration graph (for further details see text) Species Band Rm mol. wt Upogebia stellata 1 0.393 84 700 2 0.428 76 900 3 0.459 70 900 Upogebia deltaura 1 0.393 84 700 2 0.425 77 400 3 0.455 71 500 Calocaris macandreae 1 0.357 93 700 2 0.386 86 200 3 0.427 77 000 4 0.454 71 700 Callianassa subterranea 1 0.370 90 300 2 0.416 79 400 3 0.430 76 500 4 0.460 70 700 Jaxea nocturna 1 0.368 90 900 2 0.381 87 500 3 0.400 83 000 4 0.418 79 000 5 0.415 71 600

(Mr 5approx. 480 000 Da). The haemocyanin of Upogebia deltaura was also present mainly in the form of eikositetramers but with a signi®cant proportion present as dodecamers. The presence of haemocyanin in the fractions corresponding to the peaks

Fig. 3. Absorbance scans (at 280 nm) of the haemolymph samples of three of the ®ve species of thalassinidean mud-shrimps analysed by FPLC. The peaks represent the different aggregation states of the haemocyanin and indicate the presence of eikositetramers (e), dodecamers (d) or hexamers (h). A5Calocaris macandreae, B5Upogebia deltaura,C5Callianassa subterranea. 276 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 eluted from the column was con®rmed by the presence of the absorbance peak at 335 nm which disappeared almost completely when the fractions were treated with sodium sulphite. Because it was necessary to use pooled samples to obtain suf®cient haemolymph for the FPLC analyses, the analyses were repeated using pooled samples for each species obtained from different groups of animals to ensure that the results were repeatable. The absorbance scans obtained for these different samples were almost identical within each species. The effect of one freeze±thaw cycle at 2208C on the haemocyanin association of haemolymph from Calocaris macandreae was also investigated. There was little difference in the haemocyanin association state following thawing except that there was a slight decrease in the proportion of eikositetramers and a corresponding increase in the proportion of the hexamers. The addition of protease inhibitors to the haemolymph did not appear to affect the association state of the haemocyanin.

4. Discussion

4.1. Haemocyanin aggregation states

The haemocyanin molecule in many decapods exists as either hexamers or dodecam- ers with either form being predominant and the levels of native aggregates being species-speci®c (Markl, 1986). The hexamer is the typical aggregate in caridean, penaeid, pagurid and palinuran decapods (Van Holde and Miller, 1982; Mangum, 1983a). In many other decapod species, however, the haemocyanin molecules are primarily dodecamers, although in some groups both hexamers and dodecamers may co-exist in differing proportions (Markl et al., 1979; Van Holde and Miller, 1982). The haemocyanins of Calocaris macandreae and Jaxea nocturna were found to exist mainly as eikositetramers (24-mers) with a smaller amount present as hexamers. In Upogebia deltaura, however, the haemocyanin was present as a mixture or eikositetramers and dodecamers with the former being present in greater proportions. The formation of eikositetramers has previously been recorded for a number of thalassinidean shrimps; Calocaris macandreae (Svedberg, 1933), Neotrypaea (as Callianassa) californiensis (Roxby et al., 1974), N.(asCallianassa) gigas (Miller et al., 1977) and Upogebia pugettensis (Miller et al., 1977). The occurrence of eikositetramers among the thalassinidean mud-shrimps appears to be almost unique within the Crustacea but any physiological signi®cance remains to be established. The haemocyanins from these thalassinidean shrimps have been shown to exist in at least three different stable states of aggregation depending on the temperature, pH and ionic composition of the solution (Roxby et al., 1974; Miller et al., 1977; Miller and Van Holde, 1981a,b). In U. pugettensis, Miller et al. (1977) found evidence for a stable dodecamer at room temperature as was observed in U. deltaura (this study). In N.(asCallianassa) gigas, however, the dodecameric component was largely absent above 208C (Blair and Van Holde, 1976). At room temperature the haemocyanin of Callichirus (as Callianassa) major was composed primarily of eikositetramers whereas a reduction in temperature favoured the dissociation of the eikositetramer (Miller and Van Holde, 1981a). A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 277

The association of hexamers and dodecamers requires divalent cations, either Mg21 or Ca21 , as well as competent monomers (Mangum, 1983a). Van Holde et al. (1977) found that about 3 Mg21 were required per hexamer of Neotrypaea (as Callianassa) californiensis haemocyanin. The importance of Mg21 ions in the formation of eikositetramers has also been clearly demonstrated by Miller et al. (1977). It was not unexpected, therefore, to ®nd that the concentrations of Mg21 in the haemolymph were quite high (34.4±45.4 mmol l21 ) in the ®ve species of mud-shrimps examined during this study but even higher values have been measured in the haemolymph of N. californiensis (48 mmol l21 ), N. gigas (54.5 mmol l21 ) and U. pugettensis (60.0 mmol l212 ) (Miller et al., 1977). These Mg1 levels are much higher than those recorded for nearly all other decapods (Robertson, 1960; Mantel and Farmer, 1983). Although Mg21 ions are required for the formation of different aggregation states, high levels of these cations are regarded as potentially disadvantageous because of the possibility of narcotizing effects (Robertson, 1960; Mangum, 1983a).

4.2. Haemocyanin subunit structural composition

SDS PAGE studies showed that the haemocyanins of Upogebia deltaura and U. stellata consist of three major mobility groups with a large part of the haemocyanin present in a single band (MW584 700 Da). Interestingly, Miller et al. (1977) observed a similar banding pattern for U. pugettensis. The scans of the SDS±PAGE gels of Calocaris macandreae, Jaxea nocturna and Callianassa subterranea haemolymph, although not identical, exhibit a number of common components. Similarly, as many as 6 subunits were resolved for Neotrypaea (as Callianassa) californiensis and 4 subunits for N. gigas (Miller et al., 1977). Roxby et al. (1974) found that SDS±PAGE of the 24-mer of N. californiensis (termed haemocyanin C) produced only one major subunit of 70±75 000 Da. The incompetent hexamer of N. californiensis (termed haemocyanin I), as well as showing a strong band at 70±75 000, also gave 3 distinct smaller subunits (Roxby et al., 1974). Although Miller et al. (1977) did not ®nd any clear correlation between the subunit composition and the capacity to form 24-mers for U. pugettensis, N. californiensis and N. gigas, the authors did suggest that the stability of both the eikositetramer and dodecamers probably results from differences in the subunit composition. Similarly, in this study there appeared to be little correlation between the range of subunits and the ability to form aggregated structures, especially since the haemocyanin from the three species examined formed similar aggregation states. In other species, between one and eight different haemocyanin subunits have been observed (with average molecular weights ranging from 70±90 000 Da) with as many as three smaller additional non-respiratory proteins (e.g. Markl et al., 1979; Mangum, 1983a). It should be noted, however, that electrophoresis studies of crustacean haemocyanins using non-denaturing conditions frequently indicate the presence of greater numbers of bands. Such studies have demonstrated the occurrence of considerable sub-unit heterogeneity even among individuals of the same species (e.g. Markl, 1986; Markl and Decker, 1992; Reese and Mangum, 1994, Mangum and Greaves, 1996) and there is growing evidence that such 278 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 heterogeneity may in¯uence the respiratory properties of the haemocyanin (Mangum and Rainer, 1988; Mangum et al., 1991; Reese and Mangum, 1994). For example, Mangum and Rainer (1988) showed that differences in the proportions of different subunits of the haemocyanin of the crab, Callinectes sapidus collected from estuarine and from fully marine habitats resulted in signi®cantly different oxygen af®nities. Further studies of this species showed that prolonged exposure (7±25 days) to hypoxia also caused changes in the subunit composition of the haemocyanin resulting in a corresponding increase in oxygen af®nity (DeFur et al., 1990).

4.3. Oxygen transporting properties of the haemolymph

The oxygen carrying capacities (CHCYO 2 ) determined during the present study ranged from 0.22 mmol l21 for Upogebia stellata to 0.83 mmol l21 for Callianassa subterranea (Table 2), re¯ecting differences in haemocyanin concentration. These values are similar to that of 0.45 mmol l21 obtained for Neotrypaea (as Callianassa) californiensis (Miller et al., 1976), and are within the range recorded for many aquatic decapods (Mangum, 1983a). The haemocyanins of all ®ve thalassinidean species examined during the present study are characterised by having a high oxygen af®nity although the oxygen af®nity of the haemocyanin of the two upogebiids was signi®cantly lower than in the other species of mud-shrimp. These differences in P50 cannot be attributed to differences in the concentration of L-lactate or other ions in the haemolymph, since these did not differ signi®cantly between samples. These oxygen af®nities are slightly higher than those previously reported for Neotrypaea (as Callianassa) californiensis (P5052.5 at pH 8.2, 108C) (Miller and Van Holde, 1981a); N. californiensis (P5054, pH 8.0, 108C) (Miller et al., 1977); N. californiensis (P5053.7, pH 8.0, 158C) (Sanders and Childress, 1992); N. gigas (P5054, pH 8.0, 108C) (Miller et al., 1977); Upogebia pugettensis (P50511.5, pH 7.85, 108C) (Miller et al., 1977). The haemocyanin oxygen af®nities of burrowing crustaceans that regularly experience hypoxia appear to be consistently higher than those of decapods from normoxic habitats (McMahon, 1984; Taylor et al., 1985; Bridges, 1986; Taylor, 1988; Atkinson and Taylor, 1988; Sanders et al., 1988; Sanders and Childress, 1990). Among the thalassini- deans there is growing evidence to suggest that functional differences in oxygen binding characteristics are correlated with the degree of hypoxia experienced. The higher oxygen af®nity of the haemocyanin of the deposit feeding shrimps Callianassa spp., Jaxea nocturna and Calocaris macandreae (Miller et al., 1977; this study) compared with that of the haemocyanin of the ®lter feeding upogebiids indicates that the haemocyanin of the deposit feeders is particularly suited for the more severely hypoxic conditions found within their burrows. For each of the thalassinidean species, the Bohr coef®cient was moderately large (f 521.06 to 21.48), although within the range given for a number of other burrowing and non-burrowing decapod crustaceans (Mangum, 1983a; Taylor et al., 1985; Bridges, 1986). These values are similar to those obtained in previous studies of thalassinidean decapods, e.g. 21.12 for Neotrypaea (as Callianassa) californiensis (Sanders and Childress, 1992) and 21.15 for N. californiensis (Miller et al., 1977) but a A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 279 somewhat lower value of 20.63 was found for Upogebia pugettensis (Miller et al., 1977). Temperature is known to affect the oxygen af®nity of decapod haemocyanins but the magnitude of the decrease in oxygen af®nity with an increase in temperature varies greatly. There is some evidence that the haemocyanins of species that regularly experience ¯uctuations in environmental temperature show a reduced temperature sensitivity whereas other species that experience less temperature ¯uctuation have haemocyanins showing a greater temperature sensitivity (Jokumsen and Weber, 1982; Morris et al., 1985; Taylor et al., 1985). The data from this study provide some support for this theory. Calocaris macandreae were collected from sites at which the annual temperature variation is modest (temperature minima and maxima [1982±1996] 68C and 128C, Scottish Environmental Protection Agency, unpubl. data). The other species were collected from shallower waters where the annual temperature variation was greater (4±158C; D.J. Hughes, S.J. Marrs and Scottish Environmental Protection Agency, unpubl. data). As might be expected, the haemocyanin of these mud-shrimps showed a moderate temperature sensitivity. The differences in temperature sensitivity between species may have an alternative explanation, however. Burnett et al. (1988) have noted that there is some evidence of an inverse relationship between the magnitude of the Bohr effect and the temperature sensitivity of the haemocyanin. These authors suggested that when the effect of pH on oxygen af®nity is large, a low temperature sensitivity would minimize the indirect effect of temperature due to the thermal sensitivity of haemolymph pH. Whilst this may be true for some species, there are a number of species that exhibit a large Bohr effect and a large temperature sensitivity (Jokumsen et al., 1981; Mauro and Mangum, 1982; Bridges et al., 1983; Morris and Bridges, 1985, 1986). It is well established that, in addition to pH and temperature, the oxygen af®nity of decapod haemocyanins can be modulated by other factors such as organic and inorganic ions (see review by Mangum, 1983b; Morris, 1990). A number of studies have clearly demonstrated the importance of L-lactate and also urate in the modulation of haemocyanin oxygen af®nity in many, but not all, crustaceans (e.g. Truchot, 1980; Booth et al., 1982; Bridges et al., 1984; Morris et al., 1985; Lallier et al., 1987; Lallier and Truchot, 1989; Morris, 1990; Zeis et al., 1992). Although the effect of urate was not examined during the present study, no speci®c lactate effect could be demonstrated for Calocaris macandreae. Similarly, no lactate effect was observed for the haemocyanin of Neotrypaea (as Callianassa) californiensis (Mangum, 1983b). Further studies are needed to establish if this is a characteristic feature of the haemocyanins of thalassinidean mud-shrimps but there are data that indicate that the haemocyanins of some other decapods also lack a lactate effect or show a very small lactate sensitivity (Morris and Bridges, 1985, 1986; Johnson, 1987; Morris et al., 1988). The occurrence of a lactate effect is not restricted to decapods but has also been demonstrated in amphipod crustaceans (Spicer and McMahon, 1990; Spicer and Taylor, 1994). As in the decapods, however, not all species exhibit this effect. This appears to be true especially of the semi-/euterrestrial talitrid species (Spicer and McMahon, 1990; Spicer et al., 1990; Spicer and Taylor, 1994). Similarly, Morris (1991), in his review of factors affecting gas transport in decapods, recorded that over 20 species of terrestrial decapod showed no or 280 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 only a small lactate effect. The occurrence of a lactate effect in some, but not all, decapod (and amphipod) crustaceans has yet to be fully explained. It is interesting to note that studies using isothermal titration calorimetry have shown that lactate does not bind with the haemocyanin of species such as C. macandreae which do not exhibit a lactate effect, whereas in species that do, lactate binding to the haemocyanin molecule does occur (Taylor et al., unpublished data). Further work to extend the earlier studies of Graham (1985) on the mechanisms of lactate binding is required. The haemocyanins of thalassinidean mud-shrimps therefore show some differences with those of many other decapods in terms of their aggregation state and in the apparent absence of a lactate effect on haemocyanin oxygen af®nity. The high oxygen af®nity of the haemocyanin appears to be an adaptation to the hypoxic conditions that regularly occur within their burrows. It is interesting to note, however, that the upogebiid shrimps have a lower oxygen af®nity than the other species studied. Such differences between the oxygen af®nity of thalassinidean haemocyanins may re¯ect adaptations to the different feeding lifestyles and burrow environments. For example, the suspension feeding upogebiids exhibit a much more continuous pattern of burrow irrigation than the mainly deposit feeding species such as C. macandreae and Jaxea nocturna (Astall et al.,

1997). As a result, the PO2 of the burrow water rarely decreases to the very hypoxic levels recorded in the burrows of species such as C. macandreae and C. subterranea. Therefore, although the extent of burrow hypoxia varies between the species investi- gated, all have a high af®nity respiratory pigment, indicating adaptation to life in these conditions.

Acknowledgements

This work was carried out while CAA was in receipt of a NERC Research Studentship. We are grateful for the help of the crews of the research vessels Aora and Aplysia and to the dive technicians at Millport for their help in collecting animals. [SS]

References

Altman, P.L., Dittmer, D.S., 1971. Respiration and Circulation, Federation of American Society of Experimen- tal Biologists, Bethesda, Biological Handbook, Vol. 3. Arisaka, F., Van Holde, K.E., 1979. Allosteric properties and the association equilibria of hemocyanin from Callianassa californiensis. J. Mol. Biol. 134, 41±73. Astall, C.A., Taylor, A.C., Atkinson, R.J.A., 1997. Behavioural and physiological implications of a burrow- dwelling lifestyle for two species of upogebiid mud-shrimp (Crustacea: Thalassinidea). Estuar. Coastal and Shelf Sci. 44, 155±168. Atkinson, R.J.A., Taylor, A.C., 1988. Physiological ecology of burrowing decapods. In: Fincham, A.A., Rainbow, P.S. (Eds.), Aspects of the Biology of Decapod Crustacea. Symposium of the Zoological Society of London, No. 59, Oxford University Press, pp. 201±226. Blair, D.P., Van Holde, K.E., 1976. Sedimentation equilibrium studies of a complex association reaction. Biophys. Chem. 5, 165±170. A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 281

Booth, C.E., McMahon, B.R., Pinder, A.W., 1982. Oxygen uptake and the potentiating effects of increased hemolymph lactate on oxygen transport during exercise in the blue crab, Callinectes sapidus. J. Comp. Physiol. 148, 111±121. Bridges, C.R., 1986. A comparative study of the respiratory properties and physiological function of haemocyanin in two burrowing and two non-burrowing crustaceans. Comp. Biochem. Physiol. 83A, 261±270. Bridges, C.R., Bicudo, J.E.P.W., Lykkeboe, G., 1979. Oxygen content measurement in blood containing haemocyanin. Comp. Biochem. Physiol. 62A, 457±462. Bridges, C.R., Morris, S., 1986. Modulation of haemocyanin oxygen af®nity by L-lactate±a role for other cofactors. In: Linzen, B. (Ed.), Invertebrate Oxygen Carriers, Springer-Verlag, Berlin, pp. 341±352. Bridges, C.R., Morris, S., Grieshaber, M.K., 1984. Modulation of the haemocyanin oxygen af®nity of the intertidal prawn Palaemon elegans (Rathke). Respir. Physiol. 57, 189±200. Bridges, C.R., Savel, A., Stocker, W., Markl, J., Linzen, B., 1983. Structure and function of krill (Euphausia superba) haemocyanin-adaptation to life at low temperature. In: Wood, E.J. (Ed.), Structure and Function of Respiratory Proteins, Harwood Academic Publishing, pp. 353±356. Burnett, L.E., Scholnick, D.A., Mangum, C.P., 1988. Temperature sensitivity of molluscan and hemocyanins. Biol. Bull. Mar. Biol. Lab. Woods Hole 174, 153±162. DeFur, P.L., Mangum, C.P., Reese, J.P., 1990. Respiratory responses of the blue crab Callinectes sapidus to long-term hypoxia. Biol. Bull. Mar. Biol. Lab. Woods Hole 178, 46±54. Engel, P., Jones, J.B., 1978. Causes and elimination of erratic blanks in enzymatic metabolite assays involving the use of NAD1 in alkaline hydrazine buffers: improved conditions for the assay of L-glutamate, L-lactate and other metabolites. Anal. Biochem. 88, 475±484. Graham, R.A., 1985. A model for L-lactate binding to Cancer magister haemocyanin. Comp. Biochem. Physiol. 81B, 885±887. Gutmann, I., Wahlefeld, A.W., 1974. L2(1)-lactate. Determination with lactate dehydrogenase and NAD. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, Second edition, Academic Press, New York, pp. 1464±1468. Johns, A.R., Taylor, A.C., Atkinson, R.J.A., Grieshaber, M.K., 1997. Sulphide metabolism in the burrowing mud shrimp, Calocaris macandreae Bell (Crustacea: Thalassinidea). J. Mar. Biol. Ass. UK 77, 127±144. Johnson, B.A., 1987. Structure and function of the hemocyanin from a semi-terrestrial crab, Ocypode quadrata. J. Comp. Physiol. 157B, 501±509. Jokumsen, A.R., Weber, R.E., 1982. Haemocyanin oxygen af®nity in hermit crab blood is temperature independent. J. Exp. Zool. 221, 389±394. Jokumsen, A.R., Wells, R.M.G., Ellerton, H.D., Weber, R.E., 1981. Hemocyanin of the giant Antarctic isopod Glyptonotus antarcticus: structure and effects of temperature and pH on its oxygen af®nity. Comp. Biochem. Physiol. 70A, 91±95. Koike, I., Mukai, H., 1983. Oxygen and inorganic nitrogen contents and ¯uxes in burrows of the shrimps Callianassa japonica and Upogebia major. Mar. Ecol. Prog. Ser. 12, 185±190. Lallier, F., Boitel, F., 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. 86A, 255±260. Lallier, F., Truchot, J.P., 1989. Modulation of haemocyanin oxygen af®nity by L-lactate and urate in the prawn Penaeus japonicus. J. Exp. Biol. 147, 133±146. Lindahl, U., Baden, S.P., 1997. Type three functional response in ®lter feeding of the burrowing shrimp Upogebia deltaura (Leach). Ophelia 47, 33±41. McMahon, B.R., 1984. Functions and functioning of crustacean hemocyanin. In: Lamy, J.N., Truchot, J.P., Gilles, R. (Eds.), Respiratory Pigments in Animals. Relation, Structure and Function, Springer-Verlag, Berlin, pp. 35±58. Mangum, C.P., 1983. Oxygen transport in the blood. In: Mantel, L.H. (Ed.); Bliss, D.E (Series Ed.), The Biology of Crustacea. Vol. 5. Internal Anatomy and Physiological Regulation. Academic Press, New York, pp. 373±429. Mangum, C.P., 1983b. On the distribution of lactate sensitivity among hemocyanins. Mar. Biol. Lett. 4, 139±149. 282 A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283

Mangum, C.P., Greaves, J., 1996. Hemocyanins of the genus Uca: structural polymorphisms and native oligomers. J. Exp. Mar. Biol. Ecol. 199, 1±15. Mangum, C.P., Greaves, J., Rainer, J.S., 1991. Oligomer composition and oxygen binding of the hemocyanin of the blue crab Callinectes sapidus. Biol. Bull. Mar. Biol. Lab. Woods Hole 181, 453±458. Mangum, C.P., Miller, K.I., Scott, J.L., Van Holde, K.E., Morse, M.P., 1987. Bivalve hemocyanin: structural, functional and phylogenetic relationships. Biol. Bull. Mar. Biol. Lab. Woods Hole 173, 205±221.

Mangum, C.P., Rainer, J.S., 1988. The relationship between subunit composition and O2 binding of blue crab hemocyanin. Biol. Bull. Mar. Biol. Lab. Woods Hole 174, 77±82. Mantel, L.H., Farmer, L.L., 1983. Osmotic and ionic regulation. In: Mantel, L.H. (Ed.); Bliss, D.E (Series Ed.), The Biology of the Crustacea Vol. 5. Internal Anatomy and Physiological Regulation. Academic Press, New York, pp. 53±161. Markl, J., 1986. Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from . Biol. Bull. Mar. Biol. Lab. Woods Hole 171, 90±115. Markl, J., Decker, H., 1992. Molecular structure of the arthropod hemocyanins. In: Mangum, C.P. (Ed.), Blood and Tissue Oxygen Carriers, Springer-Verlag, Heidelberg, pp. 325±376. Markl, J., Decker, H., Stocker,È W., Savel, A., Linzen, B., Schutter, W.G., Van Bruggen, E.F.J., 1979. Subunit heterogeneity in arthropod hemocyanins. II. Crustacea. J. Comp. Physiol. 133, 167±175. Mauro, N.A., Mangum, C.P., 1982. The role of the blood in the temperature dependence of oxidative metabolism in decapod crustaceans, II. Interspeci®c adaptations to latitudinal changes. J. Exp. Zool. 219, 189±195. Miller, K.I., Eldred, N.W., Arisaka, F., Van Holde, K.E., 1977. Structure and function of hemocyanin from thalassinid shrimp. J. Comp. Physiol. 115, 171±184. Miller, K.I., Pritchard, A.W., Rutledge, P.S., 1976. Respiratory regulation and the role of blood in the burrowing shrimp Callianassa californiensis (: Thalassinidea). Mar. Biol. Berlin 36, 233±242. Miller, K.I., Van Holde, K.E., 1981a. The effect of environmental variables on the structure and function of hemocyanin from Callianassa californiensis I. Oxygen binding. J. Comp. Physiol. 143, 252±260. Miller, K.I., Van Holde, K.E., 1981b. The effect of environmental variables on the structure and function of hemocyanin from Callianassa californiensis II. Subunit association±dissociation equilibrium. J. Comp. Physiol. 143, 261±267. Morris, S., 1988. Effects of freezing on the function and association state of crustacean haemocyanins. J. Exp. Biol. 138, 535±539. Morris, S., 1990. Organic ions as modulators of respiratory pigment function during stress. Physiol. Zool. 62, 253±287. Morris, S., 1991. Respiratory gas exchange and transport in crustaceans: ecological determinants. Mem. Queensland Mus. 31, 241±261. Morris, S., Bridges, C.R., 1985. An investigation of haemocyanin oxygen af®nity in the semi-terrestrial crab Ocypode saratan Forsk. J. Exp. Biol. 117, 119±132. Morris, S., Bridges, C.R., 1986. Oxygen binding by the hemocyanin of the terrestrial hermit crab Coenobita clypeatus (Herbst) ± the effect of physiological parameters in vitro. Physiol. Zool. 59, 606±615. Morris, S., Greenaway, P., McMahon, B.R., 1988. Oxygen and carbon dioxide transport by the haemocyanin of an amphibious crab Holthuisana transversa. J. Comp. Physiol. 157B, 873±882. Morris, S., Taylor, A.C., Bridges, C.R., Grieshaber, M.K., 1985. Respiratory properties of the haemolymph of the intertidal prawn Palaemon elegans (Rathke). J. Exp. Zool. 233, 175±186. Nickell, L.A., Atkinson, R.J.A., 1995. Functional morphology of burrows and trophic modes of the thalassinidean shrimp species, and a new approach to the classi®cation of thalassinidean burrow morphology. Mar. Ecol. Prog. Ser. 128, 181±197. Nickell, L.A., Atkinson, R.J.A., Pinn, E.H., 1995. Morphology of thalassinidean (Crustacea: Decapoda) mouth parts and pereiopods in relation to feeding, ecology and grooming. J. Nat. Hist. 32, 733±761. Nickell, L.A., Hughes, D.J., Atkinson, R.J.A., 1996. Megafaunal bioturbation in organically enriched Scottish sea lochs. In: Eleftheriou, A., Ansell, A.D., Smith, C.J. (Eds.), Biology and Ecology of Shallow Coastal Waters. 28th European Marine Biology Symposium, Olsen and Olsen, Denmark, pp. 315±322. Pemberton, G.S., Buckley, D.E., 1976. Supershrimp: deep bioturbation in the Strait of Canso. Nova Scotia. Science 192, 790±791. A.C. Taylor et al. / J. Exp. Mar. Biol. Ecol. 244 (2000) 265 ±283 283

Pinn, E.H., Atkinson, R.J.A., Rogerson, A., 1998. Particle size selectivity and resource of partitioning in ®ve species of Thalassinidea (Crustacea: Decapoda). Mar. Ecol. Prog. Ser. 169, 243±250.

Reese, J.E., Mangum, C.P., 1994. Subunit composition and O2 binding of the crustacean hemocyanins: interspeci®c relationships. Biol. Bull. Mar. Biol. Lab. Woods Hole 87, 385±397. Robertson, J.D., 1960. Osmotic and ionic regulation. In: Waterman, T.H. (Ed.), Physiology of Crustacea, Vol. 1, Academic Press, New York, pp. 317±339. Roxby, R., Miller, K.I., Blair, D.P., Van Holde, K.E., 1974. Subunits and association equilibria of Callianassa californiensis hemocyanin. Biochemistry 13, 1662±1668. Sanders, N., Arp, A.J., Childress, J.J., 1988. Oxygen binding characteristics of the haemocyanins of two deep-sea hydrothermal vent crustaceans. Respir. Physiol. 71, 57±68. Sanders, N.K., Childress, J.J., 1990. A comparison of the respiratory function of the haemocyanins of vertically migrating and non-migrating pelagic, deep-sea oplophorid shrimps. J. Exp. Biol. 152, 167±187.

Sanders, N.K., Childress, J.J., 1992. Speci®c effects of thiosulphate and L-lactate on hemocyanin-O2 af®nity in a brachyuran hydrothermal vent crab. Mar. Biol. Berlin 113, 175±180.

Sick, H., Gersonde, K., 1969. Method for the continuous registration of O2 -binding curves of haemoproteins by means of a diffusion chamber. Analyt. Biochem. 32, 362±376. Spicer, J.I., McMahon, B.R., 1990. Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea) 1. pH, temperature and L-lactate sensitivity. J. Comp. Physiol. 160B, 195±200. Spicer, J.I., Taylor, A.C., 1994. Oxygen-binding from an ecological series of amphipod crustaceans. Mar. Biol. Berlin 120, 231±237.

Spicer, J.I., Taylor, A.C., McMahon, B.R., 1990. O2 -binding properties of haemocyanin from the sandhopper Talitrus saltator (Montagu, 1808) (Crustacea: Amphipoda). J. Exp. Mar. Biol. Ecol. 135, 213±228. Svedberg, T., 1933. Sedimentation constants, molecular weights and isoelectric points of the respiratory proteins. J. Biol. Chem. 103, 311±325. Taylor, A.C., 1988. The ecophysiology of decapods in the rock pool environment. In: Fincham, A.A., Rainbow, P.S. (Eds.), Aspects of Decapod Crustacean Biology, Symp. Zool. Soc. Lond., Vol. 59, pp. 227±261. Taylor, A.C., Morris, S., Bridges, C.R., 1985. Oxygen and carbon dioxide transporting properties of the blood of three sublittoral species of burrowing crab. J. Comp. Physiol. 155, 733±742. Terwilliger, N.B., Terwilliger, R.C., Applestein, M., Bonaventura, C., Bonaventura, J., 1979. Subunit structure and oxygen binding by hemocyanin of the isopod Ligia exotica. Biochemistry 18, 101±108. Truchot, J.P., 1976. Carbon dioxide combining properties of the blood of the shore crab Carcinus maenas (L.): carbon dioxide solubility coef®cient and carbonic acid dissociation constants. J. Exp. Biol. 64, 45±57. Truchot, J.P., 1980. Lactate increases the oxygen af®nity of crab haemocyanin. J. Exp. Zool. 214, 205±208. Tucker, V.A., 1967. Method for oxygen content and dissociation curves on microliter blood samples. J. Appl. Physiol. 23, 410±414. Van Holde, K.E., Blair, D., Eldred, N., Arisaka, F., 1977. Association equilibria of Callianassa hemocyanin. In: Banister, J.V. (Ed.), Structure and Function of Haemocyanin, Springer-Verlag, Berlin, pp. 22±30. Van Holde, K.E., Miller, K.I., 1982. Haemocyanins. Q. Rev. Biophys. 15, 1±129. Zainal, K.A.Y., Taylor, A.C., Atkinson, R.J.A., 1992. The effect of temperature and hypoxia on the respiratory physiology of the squat lobsters, Munida rugosa and Munida sarsi (Anomura Galatheidae). Comp. Biochem. Physiol. 101A, 557±567. Zeis, B., Nies, A., Bridges, C.R., Grieshaber, M.K., 1992. Allosteric modulation of haemocyanin oxygen- af®nity by L-lactate in the lobster, Homarus vulgaris. 1. Speci®c and additive effects on oxygen af®nity. J. Exp. Biol. 168, 93±110.