After exercise or periods of hypoxia, lactate accumulates in the tissues and haemolymph of crustaceans, seen here as a significant increase in lactate in both the N. puber and H. gammarus chronic exposure groups at salinity 55. Lactate is produced under these conditions as the main end product of anaerobiosis (Fincham and Rainbow 1988; Sneddon et al. 1998) leading to an acidosis of the blood evidenced by the significant decrease in pH seen in the chronic test group of N. puber. These changes at the higher salinities were not replicated in the controls, suggesting for N. puber, that the experimental set up and protocol was not responsible for the differences. This is consistent with Wyman et al (1985) who also found that handling and bleeding procedures did not cause elevated blood glucose and lactate levels in captive N. puber. The chronic control portion of the trial for H. gammarus also showed significant changes in the lactate levels over time however there was no evident linear trend, except for a massive increase on the final day of the trial. This increase in the control group may again reflect starvation or hypoxic effects rather than any handling or disturbance effects as in general in the control group, lactate levels ranged between 0 and 1 mmol and in the salinity stressed group ranged between 1 and 7 mmol.
N. puber has a high aerobic demand with the highest circulating oxygen levels and oxygen carrying capacities when compared to other sublittoral crabs such as C. pagurus and Maja squinado (Watt et al. 1999). Of these three species, haemolymph PCO2 values and lactate levels were also lowest
in N. puber, indicating high ventilation rates and a lower anaerobic component to the metabolism (Watt et al. 1999). This high aerobic demand is likely to be the reason why N. puber (and also the other two species used in this study) cease all observable activities in the highest salinities tested here. A build up of acidic metabolites is confirmed by the significant increase in lactate levels in the blood. The lactate accumulation accompanied by the significant reduction in blood pH found here as well as the additional high oxygen demand found by Watt et al (1999), suggest that N. puber is
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poorly adapted to respire anaerobically, with the switch to anaerobic respiration occurring in the range of salinity 50 – 55.
The pH of H. gammarus haemolymph showed the opposite response to that seen in N. puber, with significant increases in pH with increasing salinity, whereas N. puber showed significant decreases. In both the chronic test and control groups, the pH showed an increase with increasing salinity/time in the trial, then on the last day (day 24), a decrease in pH. Haemolymph pH is affected by acid metabolites such as lactic acid and carbon dioxide. Whereas in N. puber the significant acidification of the blood was as expected due to increases in lactate levels associated with hypoxia, the alkalisation of the blood of the lobsters is more difficult to explain when they also showed a significant increase in lactate levels which would be expected to prompt a drop in pH.
The Bohr Effect is a property of blood pigments such as haemocyanin where in the presence of CO2 and/or a decrease in pH, the oxygen affinity of the pigment decreases meaning that it binds to
oxygen with less affinity (Riggs 1988). As a product of the Bohr Effect the significant decrease in pH seen in this study for N. puber effected by the significant increase of L-Lactate in the blood, caused haemocyanin to carry oxygen to the tissues less effectively, increasing further anaerobiosis, and further decreasing the effectiveness of the haemocyanin pigment. The significant decrease of haemocyanin pigment found here for N. puber relates to a decrease in the oxygen bound haemocyanin rather than the total haemocyanin. This decrease in the O2 bound portion is
consistent with the lactic acidosis and anaerobic metabolism that has been shown above. The decrease in pH may also be the result of a build up of carbon dioxide in the tissues and bloodstream due to the closure effect exhibited by the crabs in the highest two salinities (whereby the crustaceans stop all observable behaviours and curl their legs beneath themselves, a behaviour suggested by Curtis et al (2007) that has the effect of preventing exchange across the gills), further enhancing the Bohr shift. When given time to acclimate to hypersalinity (as in the chronic exposure trial) there were no significant changes observed for haemocyanin levels in H. gammarus. However, when acutely exposed to an abrupt increase to ambient salinity, significant increases were observed in HCY levels with increasing salinity, which is in contrast to the findings for N.puber as is the significant increase in pH. Therefore it appears that salinity is having the opposite effect on H. gammarus as it does on N. puber. N. puber also appears to react differently in hypersaline conditions cf hyposaline. The results here suggest N. puber is in fact an effective regulator up to salinity 55 which is in contrast to the findings of Whiteley et al (2001) who
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suggested that N. puber lacks ion and osmoregulatory mechanisms and this is why thirty percent seawater (10 psu) had no effect on haemolymph acid–base adjustments.
Some crustaceans can increase the alkalinity of their blood to counter the Bohr shift (Taylor 1982; Hagerman and Uglow 1985; Truchot 1993) mainly through hyperventilation under hypoxic conditions. This may be an adaptation designed to counter the potential reduction in the oxygen affinity of haemocyanin caused by changes in the ionic concentration of the blood when exposed to changing salinities, therefore ensuring that under endurable salinity changes the O2 transport of the
blood can be maintained at least for a limited time. This adaptation may, under normal field conditions such as during a tidal cycle, be sufficient to help the animal survive environmental salinity fluctuations. However this adaptation was not seen here in N. puber suggesting that N. puber is poorly adapted to long term hypersaline exposure. It may be possible that the increases in pH seen for H. gammarus are indicative of a mechanism like this for compensating for acidosis. Under aerial exposure 75% of the buffering capacity of H. gammarus against haemolymph acidosis was accounted for by bicarbonate ions at 10 °C (Whiteley and Taylor 1990). In Crangon crangon haemocyanin production was increased in mild hypoxic conditions but under starvation, haemolymph haemocyanin levels decreased (Hagerman 1986), the same was found for starved H. gammarus (Hagerman 1983). These findings may explain the significant decrease in haemocyanin seen in N. puber, as in addition to acidosis of the blood causing a drop in the O2 bound portion of
haemocyanin, animals that survived to the end of the chronic experiment were not fed for 24 days (no changes in haemocyanin were found for H. gammarus and C. pagurus). However, if high environmental salinity is experienced in the field, inducing the closure response observed here in all three test species, then starvation would be likely to occur as the crabs do not move and so quickly die, there is no adaptation to salinities of 55 and above even after 96h have elapsed. This closure response induced by hypersaline exposure means there will be no aerobic respiration and no feeding; leading to a lower O2 bound portion of haemocyanin and less haemocyanin production.