A .C. Taylor et al. J. Exp. Mar. Biol. Ecol. 244 2000 265 –283
269
previously-frozen haemolymph samples were diluted to a final 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 Scientific 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 animals
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 filtration column HR10 30, Pharmacia. The column was eluted with the Ringer’s solution previously filtered through a 0.22 mm filter at a rate of 0.2
21
ml min . 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 fluoride
21
PMSF at 1 mmol l final concentration and a trypsin-like serine and cysteine
21
protease inhibitor leupeptin at 100 mmol l final concentration.
3. Results
3.1. Haemolymph ionic composition The concentrations of the major ions present in the pooled haemolymph samples of
five species of thalassinidean mud-shrimps are given in Table 1. The concentrations of
1
these ions did not differ markedly between species although the concentrations of Na
2
and Cl 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 five species of thalassinidean mud-shrimps studied. All measurements were made from pooled, frozen haemolymph samples
21
Species Concentration mmol l
1 2
1 21
21
Na Cl
K Mg
Ca 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
21
34.4–45.4 mmol 1 . The concentration of L-lactate in pooled haemolymph taken
21
from quiescent individuals was low 0.17–0.58 mmol 1 for all five 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 C
O of each of the
HCY 2
five species are given in Table 2. C . subterranea had a significantly higher p,0.05
C O
than the other mud-shrimps examined. There was no significant difference
HCY 2
p .0.05 in C O between U
. stellata, J. nocturna and C. macandreae although the
HCY 2
values for U . deltaura were significantly higher p,0.05 than in these species.
3.3. Haemocyanin oxygen affinity The relationships between pH and the log P
and between pH and n of the
50 50
haemocyanin for each of the five species are shown in Fig. 1 and Table 3. Each of the
Table 2 The oxygen-carrying of the haemocyanin C
O measured at 108C for the five species of thalassinidean
HCY 2
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[HCO ] DpH calculated from the
3
relationship between PCO and pH have been included for further details see text. All values except those
2
for buffering capacity are reported as means6S.D. The number of individual observations are shown in parentheses
Species C
O pH
L-Lactate Buffer Cap.
HCY 2
21 21
21 21
mmol l mmol l
mmol l pH unit
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 P and pH A and between n
and pH B at 108C for the haemocyanin
50 50
of the five species of mud shrimp studied. The equations of the regression lines fitted 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 coefficients for the relationships between log P
and pH at 108C for the haemolymph of the five
50
species of thalassinidean mud-shrimps presented in Fig. 3. Regression equations are given in the form of log
2
P 5a 1bpH, the coefficient of determination r is also given. All relationships were significant at
50
P ,0.05. The Bohr coefficient f is quantified by the b value. Values for calculated the oxygen affinity of the
haemocyanin P at the in vivo pH Table 2 are also given
50 2
Species a
b r
P Torr
50
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 coefficients for the relationships between log P and pH at 108C determined from oxygen
50
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 P 5a 1bpH, the
50 2
coefficient of determination r is also given. For each of the regression equations n 58
2
[L-Lactate] a
b r
21
mmol l 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 coefficient 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 significant ANCOVA, P .0.05. The oxygen affinity of the haemocyanin from all species was quite high
although there were some differences between species. In particular, the oxygen affinities of the haemocyanin of C
. subterranea, J. nocturna and C. macandreae were significantly 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 five species studied n
range52.3–3.8. For all species, pH had no significant effect on
50
the value of n Fig. 3.
50
The effect of L-lactate on the oxygen affinity 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 P and pH for the haemolymph of C
.
50
macandreae are presented in Table 4. There were no significant 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 affinity of the haemocyanin.
Values for the heat of oxygenation DH calculated for the haemocyanins of the five 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 five species of mud-shrimps
studied over the temperature ranges 5–108C and 10–158C. The P values used in the calculations were
50
derived from the regression equations fitted to the relationships between pH and log P for each species at the
50
appropriate temperature and in vivo pH
21 21
Species DH kJ mol
DH kJ mol 5–108C
10–158C 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 PCO of the haemolymph could not be measured by Astrup titration because of
2
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 PCO at
2
108C determined previously during the construction of oxygen equilibrium curves.
2
These data were used to calculate the buffering capacity of the haemolymph D[HCO ]
3 2
DpH. Values for [HCO ] were calculated using the Henderson–Hasselbalch equation
3 2
[HCO3 ] ]]]]
pH 5 pK 1 log
1
aco ? PCO
2 2
with values for pK and aco being obtained from the nomograms of Truchot 1976 at
1 2
the appropriate temperature and salinity. Regression equations describing the relation-
2
ships between [HCO ] and pH were then calculated for each of the five species and the
3
values for the buffering capacity of the haemolymph obtained from the slopes of the regression equations. Since the values for the constants pK
and aco
were not
1 2
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 five 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 five 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 M 5approx. 1 900 000 Da with some hexameric haemocyanin also present
r
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, E5 Jaxea nocturna. The individual traces have been aligned against the same R
scale.
m
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 five 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
M 5approx. 480 000 Da. The haemocyanin of Upogebia deltaura was also present
r
mainly in the form of eikositetramers but with a significant 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 five 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 confirmed 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 sufficient 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
